Monomers and oligonucleotides comprising cycloaddition adduct(s)

ABSTRACT

The invention features compounds of formula V or XII: 
     
       
         
         
             
             
         
       
     
     In one embodiment, the invention relates compounds and processes for conjugating ligand to oligonucleotide. The invention further relates to methods for treating various disorders and diseases such as viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies and immunosuppression.

PRIORITY CLAIM

This application is divisional application of U.S. patent applicationSer. No. 13/575,461, filed Oct. 17, 2012, which claims priority to PCTApplication No. PCT/US2011/022975, filed Jan. 28, 2011, which claimspriority to U.S. Provisional Application No. 61/405,980, filed Oct. 22,2010 and U.S. Provisional Application No. 61/299,296, filed Jan. 28,2010, all of which are hereby incorporated by reference in theirentirety.

FIELD OF INVENTION

The present invention relates to the field of conjugation of ligands tooligonucleotides with copper free cycloaddition chemistry.

BACKGROUND

Oligonucleotide compounds have important therapeutic applications inmedicine. Oligonucleotides can be used to silence genes that areresponsible for a particular disease. Gene-silencing prevents formationof a protein by inhibiting translation. Importantly, gene-silencingagents are a promising alternative to traditional small, organiccompounds that inhibit the function of the protein linked to thedisease. siRNA, antisense RNA, and micro-RNA are oligonucleotides thatprevent the formation of proteins by gene-silencing.

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression (Fire et al. (1998) Nature 391, 806-811;Elbashir et al. (2001) Genes Dev. 15, 188-200). Short dsRNA directsgene-specific, post-transcriptional silencing in many organisms,including vertebrates, and has provided a new tool for studying genefunction. RNAi is mediated by RNA-induced silencing complex (RISC), asequence-specific, multi-component nuclease that destroys messenger RNAshomologous to the silencing trigger. RISC is known to contain short RNAs(approximately 22 nucleotides) derived from the double-stranded RNAtrigger, but the protein components of this activity remained unknown.

siRNA compounds are promising agents for a variety of diagnostic andtherapeutic purposes. siRNA compounds can be used to identify thefunction of a gene. In addition, siRNA compounds offer enormouspotential as a new type of pharmaceutical agent which acts by silencingdisease-causing genes. Research is currently underway to developinterference RNA therapeutic agents for the treatment of many diseasesincluding central-nervous-system diseases, inflammatory diseases,metabolic disorders, oncology, infectious diseases, and ocular disease.

Despite the different synthetic strategies developed for conjugation ofvarious ligands to the oligonucleotides, the synthesis ofligand-oligonucleotide conjugates is anything but trivial and requiresextensive expertise in organic chemistry and solid-phase synthesis. Areal advance would be to use a coupling reaction that can be utilizedfor a large variety of ligands and oligonucleotides. The Huisgen1,3-dipolar cycloaddition of alkynes and azides, the “click” reaction,is especially attractive for irreversible coupling of two moleculesunder mild conditions. The “click” chemistry has recently emerged as anefficient strategy to conjugate carbohydrates, peptides and proteins,fluorescent labels and lipids to oligonucleotides. Therefore, there is aclear need for new reagents that can be utilize for “click” chemistryfor conjugation of ligands to oligonucleotides. The present invention isdirected to this very important end.

SUMMARY

The invention relates to compounds that can be used as a ribosereplacement or can be used as universal base to conjugate variousligands to oligonucleotides, e.g. iRNA agents, through “copper freeclick” chemistry. These compounds are also referred to as the“click-carrier” herein.

In one aspect, the invention features, a compound having the structureshown in formula (I)

wherein:

A is O, S, NR^(N) or CR^(P) ₂;

B is independently for each occurrence hydrogen, optionally substitutednatural or non-natural nucleobase, optionally substituted triazole,optionally substituted tetrazole, R⁶, NH—C(O)—O—C(CH₂B₁)₃,NH—C(O)—NH—C(CH₂B₁)₃, where B₁ is halogen, mesylate, optionallysubstituted triazole, optionally substituted tetrazole, or R⁶;

R¹, R², R³, R⁴ and R⁵ are each independently for each occurrence H, OR⁷,F, N(R^(N))₂, N₃, CN, -J-linker-N₃, -J-linker-CN, -J-linker-R⁸,-J-linker-cycloalkyne, -J-linker-R_(L), -J-Q-linker-R^(L) or-J-linker-Q-linker-R^(L);

R³′ is H or OH;

R^(5′) is independently for each occurrence H, halogen, optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted alkynyl,

J is absent, O, S, NR^(N), OC(O)NH, NHC(O)O, C(O)NH, NHC(O), NHSO,NHSO₂, NHSO₂NH, OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH,O—N═CH, OP(N(R^(N))₂)O, or OP(N(R^(N))₂);

R⁷ is independently for each occurrence hydrogen, hydroxyl protectinggroup, optionally substituted alkyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted alkenyl, optionally substituted heteroaryl,polyethyleneglycol (PEG), a phosphate, a diphosphate, a triphosphate, aphosphonate, a phosphonothioate, a phosphonodithioate, aphosphorothioate, a phosphorothiolate, a phosphorodithioate, aphosphorothiolothionate, a phosphodiester, a phosphotriester, anactivated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z⁴)(Z⁵)—O-oligonucleotide, —P(Z⁴)(Z⁵)-formula (I),—P(Z⁴)(O-linker-Q-linker-R^(L))—O-nucleoside,—P(Z⁴)(O-linker-N₃)—O-nucleoside, P(Z⁴)(O-linker-CN)—O-nucleoside,P(Z⁴)(O-linker-R⁸)—O-nucleoside,P(Z⁴)(O-linker-cycloalkyne)-O-nucleoside,—P(Z⁴)(O-linker-R^(L))—O-oligonucleotide,—P(Z⁴)(O-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z⁴)(O-linker-R^(L))—O-oligonucleotide,P(Z⁴)(O-linker-N₃)—O-oligonucleotide,—P(Z⁴)(O-linker-CN)—O-oligonucleotide,P(Z⁴)(O-linker-R⁸)—O-oligonucleotide,P(Z⁴)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z⁴)(-linker-Q-linker-R^(L))—O-nucleoside,P(Z⁴)(-linker-R^(L))—O-nucleoside, —P(Z⁴)(-linker-N₃)—O-nucleoside,P(Z⁴)(-linker-CN)—O-nucleoside, P(Z⁴)(-linker-R⁸)—O-nucleoside,P(Z⁴)(-linker-cycloalkyne)-O-nucleoside,—P(Z⁴)(-linker-Q-linker-R^(L))—O-oligonucleotide,(Z⁴)(-linker-R^(L))—O-oligonucleotide,P(Z⁴)(-linker-N₃)—O-oligonucleotide,—P(Z⁴)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-R⁸)—O-oligonucleotide orP(Z⁴)(-linker-cycloalkyne)-O-oligonucleotide;

R⁸ is

X^(L) and Y^(L) are independently absent, a linker, —(CH₂)_(n)O—,—(CH₂)_(n)COO—, —(CH₂)_(n)N(R⁹)—, —(CH₂)_(n)S—, (CH₂)_(n)S—S—,—(CH₂)_(n)O—N(R⁹)—, or —(CH₂)_(n)CO—;

X and Y are independently H, a bond, (CH₂)_(n)OH, (CH₂)_(n)COOH,(CH₂)_(n)N(R⁹)(R⁹), (CH₂)_(n)SH, (CH₂)_(n)S—SP-Py, (CH₂)_(n)O—N(R⁹)(R⁹),(CH₂)_(n)CHO, or (CH₂)_(n)COR¹⁰;

Q¹, Q² and Q³ are independently C(R^(P))₂, NR⁹, O, or S;

Q⁴, Q⁵, Q⁶ and Q⁷ are independently CR^(P) or N;

R⁹, R¹⁰ and R^(N) are independently for each occurrence H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcycloalkyl, optionally substituted aralkyl, optionally substitutedheteroaryl or an amino protecting group;

R^(L) is hydrogen or a ligand;

R^(P) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted aralkyl or optionally substituted heteroaryl;

Q is independently for each occurrence

Y₁, Y₂ and Y₃ are independently CR^(P), N, O, or S;

Z¹, Z² and Z³ are independently H or R⁸;

Z⁴ and Z⁵ are each independently for each occurrence O, S or optionallysubstituted alkyl;

n is 0-20; and

provided that at least one R⁸ or Q is present.

In one embodiment, the invention features, a compound having thestructure shown in formula (II)

A and B are independently for each occurrence hydrogen, protectinggroup, optionally substituted aliphatic, optionally substituted aryl,optionally substituted heteroaryl, polyethyleneglycol (PEG), aphosphate, a diphosphate, a triphosphate, a phosphonate, aphosphonothioate, a phosphonodithioate, a phosphorothioate, aphosphorothiolate, a phosphorodithioate, a phosphorothiolothionate, aphosphodiester, a phosphotriester, an activated phosphate group, anactivated phosphite group, a phosphoramidite, a solid support,—P(Z⁴)(Z⁵)—O-nucleoside, or —P(Z⁴)(Z⁵)—O-oligonucleotide; wherein Z⁴ andZ⁵ are each independently for each occurrence O, S or optionallysubstituted alkyl;

J₁ and J₂ are independently O, S, NR^(N), optionally substituted alkyl,OC(O)NH, NHC(O)O, C(O)NH, NHC(O), OC(O), C(O)O, OC(O)O, NHC(O)NH,NHC(S)NH, OC(S)NH, OP(N(R^(P))₂)O, or OP(N(R^(P))₂);

is cyclic group or acyclic group; preferably, the cyclic group isselected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl,isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl,quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably,the acyclic group is selected from serinol backbone or diethanolaminebackbone;

L₁₀ and L₁₁ are independently absent or a linker;

Q is independently for each occurrence

X^(L) and Y^(L) are independently absent, a linker, —(CH₂)_(n)O—,—(CH₂)_(n)COO—, —(CH₂)_(n)N(R⁹)—, —(CH₂)_(n)S—, (CH₂)_(n)S—S—,—(CH₂)_(n)O—N(R⁹)—, or —(CH₂)_(n)CO—;

X and Y are independently H, a bond, (CH₂)_(n)OH, (CH₂)_(n)COOH,(CH₂)_(n)N(R⁹)(R⁹), (CH₂)_(n)SH, (CH₂)_(n)S—SP-Py, (CH₂)_(n)O—N(R⁹)(R⁹),(CH₂)_(n)CHO, or (CH₂)_(n)COR¹⁰;

R⁹, and R¹⁰ are independently for each occurrence H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcycloalkyl, optionally substituted aralkyl, optionally substitutedheteroaryl or an amino protecting group;

Y₁, Y₂ and Y₃ are independently CR^(P), N, O, or S;

R^(P) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted aralkyl or optionally substituted heteroaryl;

Q¹, Q² and Q³ are independently C(R^(P))₂, NR⁹, O, or S;

Q⁴, Q⁵, Q⁶ and Q⁷ are independently CR^(P) or N;

Z⁴ and Z⁵ are each independently for each occurrence O, S or optionallysubstituted alkyl; and n is 0-20.

In another embodiment of the present invention there are disclosedpharmaceutical compositions comprising a therapeutically effectiveamount of an iRNA agent of the invention in combination with apharmaceutically acceptable carrier or excipient. In yet anotherembodiment of the invention describes process for preparing saidcompounds

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the click 5′-alkyne-RNA (A53215.1) with different azides(protected and unprotected) in solution phase. FIG. 1 discloses SEQ IDNO: 32.

FIG. 2 depicts the monitoring of reaction progress by HPLC analysis:click 5′-alkyne-RNA (A53215.1) with (a) GalNAc (protected) azide, (b)mannose (protected) azide; (c) C18 azidel in solution phase; and (d)time-course product percentage of click reactions.

FIG. 3 depicts the monitoring of reaction progress by HPLC analysis:click 5′-alkyne-RNA (A53215.1) with (a) GalNAc3 (unprotected) azide; (b)mannose (unprotected) azide in solution phase; and (c) time-courseproduct percentage of click reactions.

FIG. 4 depicts the click 3′-alkyne-RNA (A53213.1) with differentprotected azides in solution phase. FIG. 4 discloses SEQ ID NO: 33.

FIG. 5 depicts an HPLC analysis of click reactions of 3′-alkyne-RNA(A53213.1) with (a) GalNAc (protected) azide; (b) mannose (protected)azide; and (c) C18 azide in solution phase.

FIG. 5 discloses SEQ ID NO: 33.

FIG. 6 depicts the click internal-alkyne-RNA (A53214.1) with differentprotected azides in solution phase. FIG. 6 discloses SEQ ID NO: 34.

FIG. 7 depicts an HPLC analysis of click reactions ofinternal-alkyne-RNA (A53214.1) with (a) GalNAc (protected) azide; (b)mannose (protected) azide; and (c) C18 azide in solution phase. FIG. 7discloses SEQ ID NO: 34.

FIG. 8 depicts the click 5′-alkyne-RNA with GalNAc (protected) and C18azides on CPG. FIG. 8 discloses SEQ ID NO: 32.

FIG. 9 depicts an HPLC analysis of products from click reactions of5′-alkyne-RNA (A53215.1 (SEQ ID NO: 32)) with GalNAc (protected) and C18azides on CPG.

FIG. 10 depicts various 3′ and 5′ RNA conjugates with alkynederivatives.

FIG. 11 depicts some of the exemplary strained/activated alkynes.

FIG. 12 depicts nucleosides functionalized with strained/activatedalkynes for incorporation into nucleic acids. Each of Z¹, Z², and Z³comprises either an strained/activated alkyne (e.g. R⁸) or an azide,provided that when an alkyne is present the azide is not present in thesame nucleoside and when an azide is present the alkyne is not presentin the same nucleoside. The activated alkyne moiety reacts with azidofunctionalized ligand or molecules of interest to obtain the desiredconjugate.

FIGS. 13 and 14 depict some embodiments of R⁸.

FIGS. 15 and 16 depict some embodiments of Q.

DETAILED DESCRIPTION

In one embodiment of the compounds of the present invention arecompounds represented by formula I or II as illustrated above, or apharmaceutically acceptable salt, ester or prodrug thereof.

In some embodiments of the compounds described herein, R⁸ is as shown inFIGS. 13 and 14.

In some embodiments of the compounds described herein, Q is as shown inFIGS. 15 and 16.

In some embodiments of the compounds described herein Y₁, Y₂ and Y₃ areN.

In one embodiment, the invention features a compound having thestructure shown in formula (III):

wherein each linker can be the same or different, and R², R³, R⁵, R^(L)and Q are as defined for the first embodiment.

In one embodiment the invention features a compound having the structureshown in (IIIa) or (IIIb):

wherein R², R³, R⁵, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, Y, Y₁, Y₂,and Y₃ are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (IVa) or (IVb):

wherein R², R³, R⁵, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X and Y, areas defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (V):

wherein each linker can be the same or different, and B, R³, R⁵, R^(L),and Q are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (Va) or (Vb):

wherein B, R³, R⁵, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, Y, Y₁, Y₂,and Y₃ are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (VIa) or (VIb):

wherein B, R³, R⁵, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, Y, Y₁, Y₂,and Y₃ are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (VII):

wherein each linker can be the same or different, and B, R², R⁵, R^(L),and Q are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (VIIa) or (VIIb):

wherein B, R², R⁵, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, Y, Y₁, Y₂,and Y₃ are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (VIIIa) or (VIIIb):

wherein B, R², R⁵, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, and Y are asdefined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (IX):

wherein B, R², R³, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, Y, Y₁, Y₂,and Y₃ are as defined in the first embodiment. In one embodiment, theinvention features, a compound having the structure shown in formula(IXa) or (IXb):

wherein B, R², R³, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, Y, Y₁, Y₂,and Y₃ are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (Xa) or (Xb):

wherein B, R², R³, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X and Y are asdefined in the first embodiment.

In one embodiment, the carrier may be based on the pyrroline ring systemas shown in formula (XI):

wherein E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, orSO₂NH;

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are each independently foreach occurrence H, —CH₂OR^(a), or OR^(b),

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z⁴)(Z⁵)—O-oligonucleotide, —P(Z⁴)(Z⁵)-formula (I),—P(Z⁴)(O-linker-Q-linker-R^(L))—O-nucleoside,—P(Z⁴)(O-linker-N₃)—O-nucleoside, P(Z⁴)(O-linker-CN)—O-nucleoside,P(Z⁴)(O-linker-R⁸)—O-nucleoside,P(Z⁴)(O-linker-cycloalkyne)-O-nucleoside,—P(Z⁴)(O-linker-R^(L))—O-oligonucleotide,—P(Z⁴)(O-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z⁴)(O-linker-R^(L))—O-oligonucleotide,P(Z⁴)(O-linker-N₃)—O-oligonucleotide,—P(Z⁴)(O-linker-CN)—O-oligonucleotide,P(Z⁴)(O-linker-R⁸)—O-oligonucleotide,P(Z⁴)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z⁴)(-linker-Q-linker-R^(L))—O-nucleoside,P(Z⁴)(-linker-R^(L))—O-nucleoside, —P(Z⁴)(-linker-N₃)—O-nucleoside,P(Z⁴)(-linker-CN)—O-nucleoside, P(Z⁴)(-linker-R⁸)—O-nucleoside,P(Z⁴)(-linker-cycloalkyne)-O-nucleoside,—P(Z⁴)(-linker-Q-linker-R^(L))—O-oligonucleotide,(Z⁴)(-linker-R^(L))—O-oligonucleotide,P(Z⁴)(-linker-N₃)—O-oligonucleotide,—P(Z⁴)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-R⁸)—O-oligonucleotide orP(Z⁴)(-linker-cycloalkyne)-O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-Q-linker-R^(L),-linker-R^(L) or R³¹;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, -linker-Q-linker-R^(L),-linker-R^(L) or R³¹;

R^(L) is hydrogen or a ligand;

R^(N) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted aralkyl, optionally substituted heteroaryl or anamino protecting group;

R^(P) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl oroptionally substituted heteroaryl;

R⁸ is

X^(L) and Y^(L) are independently absent, —(CH₂)_(n)O—, —(CH₂)_(n)COO—,—(CH₂)_(n)N(R⁹)—, —(CH₂)_(n)S—, (CH₂)_(n)S—S—, —(CH₂)_(n)O—N(R⁹)—, or—(CH₂)_(n)CO—;

X and Y are independently H, a bond, (CH₂)_(n)OH, (CH₂)_(n)COOH,(CH₂)_(n)N(R⁹)(R⁹), (CH₂)_(n)SH, (CH₂)_(n)S—SP-Py, (CH₂)_(n)O—N(R⁹)(R⁹),(CH₂)_(n)CHO, or (CH₂)_(n)COR¹⁰;

Q¹, Q² and Q³ are independently C(R^(P))₂, NR⁹, O, or S;

Q⁴, Q⁵, Q⁶ and Q⁷ are independently CR^(P) or N;

Q is independently for each occurrence

Y₁, Y₂ and Y₃ are independently CR^(P), N, O, or S;

Z⁴ and Z⁵ are each independently for each occurrence O, S or optionallysubstituted alkyl;

R⁹, and R¹⁰ are independently for each occurrence H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcycloalkyl, optionally substituted aralkyl, optionally substitutedheteroaryl or an amino protecting group;

f and h are independently for each occurrence 1-20;

n is 0-20; and

provided that at least one R⁸ or Q is present.

For the pyrroline-based click-carriers, R¹¹ is —CH₂OR^(a) and R¹³ isOR^(b); or R¹¹ is —CH₂OR^(a) and R¹⁵ is OR^(b); or R¹¹ is —CH₂OR^(a) andR¹⁷ is OR^(b); or R¹³ is —CH₂OR^(a) and R¹¹ is OR^(b); or R¹³ is—CH₂OR^(a) and R¹⁵ is OR^(b); or R¹³ is —CH₂OR^(a) and R¹⁷ is OR^(b). Incertain embodiments, CH₂OR^(a) and OR^(b) may be geminally substituted.For the 4-hydroxyproline-based carriers, R¹¹ is —CH₂OR^(a) and R¹⁷ isOR^(b). The pyrroline- and 4-hydroxyproline-based compounds maytherefore contain linkages (e.g., carbon-carbon bonds) wherein bondrotation is restricted about that particular linkage, e.g. restrictionresulting from the presence of a ring. Thus, CH₂OR^(a) and OR^(b) may becis or trans with respect to one another in any of the pairingsdelineated above Accordingly, all cis/trans isomers are expresslyincluded. The compounds may also contain one or more asymmetric centersand thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures. All such isomericforms of the compounds are expressly included (e.g., the centers bearingCH₂OR^(a) and OR^(b) can both have the R configuration; or both have theS configuration; or one center can have the R configuration and theother center can have the S configuration and vice versa).

In one embodiment, R¹¹ is CH₂OR^(a) and R¹⁷ is OR^(b).

In one embodiment, R^(b) is a solid support.

In one preferred embodiment, R³¹ is —C(O)CH(N(R³²)₂)(CH₂)₄N(R³²)₂ and atleast one R³² is —C(O)(CH₂)_(f)CR⁸ or -linker-Q-linker-R^(L) and R^(L)is present.

In one preferred embodiment, R³¹ is —C(O)CH(N(R³²)₂)(CH₂)₄NH₂ and atleast one R³² is —C(O)(CH₂)_(f)CR⁸ or -linker-Q-linker-R^(L) and R^(L)is present.

In one embodiment, the invention features, a compound having thestructure shown in formula (XII):

wherein R_(a) and R_(b) are independently hydrogen, an activatedphosphate group, an activated phosphite group, a phosphoramidite, asolid support, —P(Z⁴)(Z⁵)—OH, —P(Z⁵)(Z⁵)—O-nucleoside,—P(Z⁵)(Z⁵)—O-oligonucleotide; each linker can be the same or different;and E, R^(L), and Q are as defined in the previous embodiments.

In one embodiment, the invention features, a compound having thestructure shown in formula (XIIa) or (XIIb):

wherein R_(a) and R_(b) are independently hydrogen, an activatedphosphate group, an activated phosphite group, a phosphoramidite, asolid support, —P(Z⁴)(Z⁵)—OH, —P(Z⁵)(Z⁵)—O-nucleoside,—P(Z⁵)(Z⁵)—O-oligonucleotide; each linker can be the same or different;and E, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, Y, Y₁, Y₂, and Y₃ are asdefined in the previous embodiments.

In one embodiment, the invention features, a compound having thestructure shown in formula (XIIIa) or (XIIIb):

wherein R_(a) and R_(b) are independently hydrogen, an activatedphosphate group, an activated phosphite group, a phosphoramidite, asolid support, —P(Z⁴)(Z⁵)—OH, —P(Z⁵)(Z⁵)—O-nucleoside,—P(Z⁵)(Z⁵)—O-oligonucleotide; each linker can be the same or different;and E, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, and Y are as defined inthe previous embodiments.

In one embodiment features acyclic sugar replacement-based compounds,e.g., sugar replacement based click-carrier compounds, are also referredto herein as ribose replacement compound subunit (RRMS) compoundcompounds. Preferred acyclic carriers can have the structure shown informula (XIV) below.

In one aspect, the invention features, an acyclic click-carrier compoundhaving the structure shown in formula (XIV)

wherein:

W is absent, O, S and N(R^(N)), where R^(N) is independently for eachoccurrence H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted heteroaryl or an amino protecting group;

E is absent or C(O), C(O)O, C(O)NH, C(S), C(S)NH, SO, SO₂, or SO₂NH;

R^(a) and R^(b) are each independently for each occurrence hydrogen,hydroxyl protecting group, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted cycloalkyl, optionallysubstituted aralkyl, optionally substituted alkenyl, optionallysubstituted heteroaryl, polyethyleneglycol (PEG), a phosphate, adiphosphate, a triphosphate, a phosphonate, a phosphonothioate, aphosphonodithioate, a phosphorothioate, a phosphorothiolate, aphosphorodithioate, a phosphorothiolothionate, a phosphodiester, aphosphotriester, an activated phosphate group, an activated phosphitegroup, a phosphoramidite, a solid support, —P(Z⁵)(Z⁵)—O-nucleoside,—P(Z⁴)(Z⁵)—O-oligonucleotide, —P(Z⁵)(Z⁵)-formula (I),—P(Z⁵)(O-linker-Q-linker-R^(L))—O-nucleoside,—P(Z⁵)(O-linker-N₃)—O-nucleoside, P(Z⁴)(O-linker-CN)—O-nucleoside,P(Z⁴)(O-linker-R⁸)—O-nucleoside,P(Z⁴)(O-linker-cycloalkyne)-O-nucleoside,—P(Z⁴)(O-linker-R^(L))—O-oligonucleotide,—P(Z⁴)(O-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z⁴)(O-linker-R^(L))—O-oligonucleotide,P(Z⁴)(O-linker-N₃)—O-oligonucleotide,—P(Z⁴)(O-linker-CN)—O-oligonucleotide,P(Z¹)(O-linker-R⁸)—O-oligonucleotide,P(Z⁴)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z⁴)(-linker-Q-linker-R^(L))—O-nucleoside,P(Z⁴)(-linker-R^(L))—O-nucleoside, —P(Z⁴)(-linker-N₃)—O-nucleoside,P(Z⁴)(-linker-CN)—O-nucleoside, P(Z⁴)(-linker-R⁸)—O-nucleoside,P(Z⁴)(-linker-cycloalkyne)-O-nucleoside,—P(Z⁴)(-linker-Q-linker-R^(L))—O-oligonucleotide,(Z⁴)(-linker-R^(L))—O-oligonucleotide,P(Z⁴)(-linker-N₃)—O-oligonucleotide,—P(Z⁴)(-linker-CN)—O-oligonucleotide,P(Z⁴)(-linker-R⁸)—O-oligonucleotide orP(Z⁴)(-linker-cycloalkyne)-O-oligonucleotide;

R³⁰ is independently for each occurrence -linker-Q-linker-R^(L),-linker-R^(L) or R³¹;

R³¹ is —C(O)CH(N(R³²)₂)(CH₂)_(h)N(R³²)₂;

R³² is independently for each occurrence H, -linker-Q-linker-R^(L),-linker-R^(L) or R³¹;

R^(L) is hydrogen or a ligand;

R^(N) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted aralkyl, optionally substituted heteroaryl or anamino protecting group;

R^(P) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl oroptionally substituted heteroaryl;

R⁸ is

X^(L) and Y^(L) are independently absent, —(CH₂)_(n)O—, —(CH₂)_(n)COO—,—(CH₂)_(n)N(R⁹)—, —(CH₂)_(n)S—, (CH₂)_(n)S—S—, —(CH₂)_(n)O—N(R⁹)—, or—(CH₂)_(n)CO—;

X and Y are independently H, a bond, (CH₂)_(n)OH, (CH₂)_(n)COOH,(CH₂)_(n)N(R⁹)(R⁹), (CH₂)_(n)SH, (CH₂)_(n)S—SP-Py, (CH₂)_(n)O—N(R⁹)(R⁹),(CH₂)_(n)CHO, or (CH₂)_(n)COR¹⁰;

Q¹, Q² and Q³ are independently C(R^(P))₂, NR⁹, O, or S;

Q⁴, Q⁵, Q⁶ and Q⁷ are independently CR^(P) or N;

Q is independently for each occurrence

Y₁, Y₂ and Y₃ are independently CR^(P), N, O, or S;

Z⁴ and Z⁵ are each independently for each occurrence O, S or optionallysubstituted alkyl;

R⁹, and R¹⁰ are independently for each occurrence H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcycloalkyl, optionally substituted aralkyl, optionally substitutedheteroaryl or an amino protecting group;

f and h are independently for each occurrence 1-20;

n is 0-20; and

provided that at least one R⁸ or Q is present

When r and s are different, then the tertiary carbon can be either the Ror S configuration. In preferred embodiments, x and y are one and z iszero (e.g. carrier is based on serinol). The acyclic carriers canoptionally be substituted, e.g. with hydroxy, alkoxy, perhaloalky.

Other carrier compounds amenable to the invention are described incopending applications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004;U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/985,426,filed Nov. 9, 2004; U.S. Ser. No. 10/833,934, filed Aug. 3, 2007; U.S.Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser. No. 11/119,533,filed Apr. 29, 2005, which are incorporated by reference in theirentireties for all purposes.

In one embodiment, at least one R¹, R², R³, R⁴, or R⁵ of formula (I) is

wherein R^(L), Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, and Y are as previouslydefined.

In one embodiment, the invention features, a compound having thestructure shown in formula (XV):

wherein each linker can be the same or different, and B, R², R³, R⁵,R^(L), and Q are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (XVa) or (XVb):

wherein B, R², R³, R⁵, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, Y, Y₁,Y₂, and Y₃ are as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (XVIa) or (XVIb):

wherein B, R², R³, R⁵, R^(L), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, and Yare as defined in the first embodiment.

In one embodiment, the invention features, a compound having thestructure shown in formula (XVIIa) or (XVIIb):

wherein R², R³, R⁵, R^(L), R^(P), J, Q₁, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X, andY are as defined in the first embodiment.

In one embodiment, the invention features a compound of formula (XVIII):

wherein Q₁, Q₂, Q₂, Q₃, Q₄, Q₅, Q₆, Q₇, X and Y are as defined in thefirst embodiment, provided that when X is H then Y is not H, and when Yis H then X is not H.

In the compounds of formula (XVIII), X and/or Y can be used forconjugation to nucleic acid (DNA, RNA, nucleic acid therapeutics such assiRNA and antisense oligonucleotides), any ligand of interest, any drugcarrier or drug delivery vehicle.

In some embodiments, a compound of formula (XVIII) is as shown in FIG.11.

In one embodiment, the invention features a compound having thestructure shown in formula (XIX)

wherein:

A is O, S, NR^(N) or CR^(P) ₂;

B is independently for each occurrence hydrogen, optionally substitutednatural or non-natural nucleobase, optionally substituted triazole,optionally substituted tetrazole, R⁶, NH—C(O)—O—C(CH₂B₁)₃,NH—C(O)—NH—C(CH₂B₁)₃, where B₁ is halogen, mesylate, optionallysubstituted triazole, optionally substituted tetrazole, or R⁶;

R²⁰, R²¹, R²², and R²³ are each independently for each occurrence H,OR⁷, F, N(R^(N))₂, N₃, CN, -J-linker-N₃, -J-linker-CN, -J-linker-R⁸,-J-linker-cycloalkyne, -J-linker-R_(L), -J-Q-linker-R^(L) or-J-linker-Q-linker-R^(L);

R²⁴ is independently for each occurrence H, halogen, optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted alkynyl,

J is absent, O, S, NR^(N), OC(O)NH, NHC(O)O, C(O)NH, NHC(O), NHSO,NHSO₂, NHSO₂NH, OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH, OC(S)NH,O—N═CH, OP(N(R^(N))₂)O, or OP(N(R^(N))₂);

R⁷ is independently for each occurrence hydrogen, hydroxyl protectinggroup, optionally substituted alkyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted alkenyl, optionally substituted heteroaryl,polyethyleneglycol (PEG), a phosphate, a diphosphate, a triphosphate, aphosphonate, a phosphonothioate, a phosphonodithioate, aphosphorothioate, a phosphorothiolate, a phosphorodithioate, aphosphorothiolothionate, a phosphodiester, a phosphotriester, anactivated phosphate group, an activated phosphite group, aphosphoramidite, a solid support, —P(Z¹)(Z²)—O-nucleoside,—P(Z⁴)(Z⁵)—O-oligonucleotide, —P(Z⁴)(Z⁵)-formula (I),—P(Z⁴)(O-linker-Q-linker-R^(L))—O-nucleoside,—P(Z⁴)(O-linker-N₃)—O-nucleoside, P(Z⁴)(O-linker-CN)—O-nucleoside,P(Z⁴)(O-linker-R⁸)—O-nucleoside,P(Z⁴)(O-linker-cycloalkyne)-O-nucleoside,—P(Z⁴)(O-linker-R^(L))—O-oligonucleotide,—P(Z⁴)(O-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z⁴)(O-linker-R^(L))—O-oligonucleotide,P(Z⁴)(O-linker-N₃)—O-oligonucleotide,—P(Z⁴)(O-linker-CN)—O-oligonucleotide,P(Z⁴)(O-linker-R⁸)—O-oligonucleotide,P(Z⁴)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z⁴)(-linker-Q-linker-R^(L))—O-nucleoside,P(Z⁴)(-linker-R^(L))—O-nucleoside, —P(Z⁴)(-linker-N₃)—O-nucleoside,P(Z⁴)(-linker-CN)—O-nucleoside, P(Z⁴)(-linker-R⁸)—O-nucleoside,P(Z⁴)(-linker-cycloalkyne)-O-nucleoside,—P(Z⁴)(-linker-Q-linker-R^(L))—O-oligonucleotide,(Z⁴)(-linker-R^(L))—O-oligonucleotide,P(Z⁴)(-linker-N₃)—O-oligonucleotide,—P(Z⁴)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-R⁸)—O-oligonucleotide orP(Z⁴)(-linker-cycloalkyne)-O-oligonucleotide;

R⁸ is

X^(L) and Y^(L) are independently absent, a linker, —(CH₂)_(n)O—,—(CH₂)_(n)COO—, —(CH₂)_(n)N(R⁹)—, —(CH₂)_(n)S—, (CH₂)_(n)S—S—,—(CH₂)_(n)O—N(R⁹)—, or —(CH₂)_(n)CO—;

X and Y are independently H, a bond, (CH₂)_(n)OH, (CH₂)_(n)COOH,(CH₂)_(n)N(R⁹)(R⁹), (CH₂)_(n)SH, (CH₂)_(n)S—SP-Py, (CH₂)_(n)O—N(R⁹)(R⁹),(CH₂)_(n)CHO, or (CH₂)_(n)COR¹⁰;

Q¹, Q² and Q³ are independently C(R^(P))₂, NR⁹, O, or S;

Q⁴, Q⁵, Q⁶ and Q⁷ are independently CR^(P) or N;

R⁹, R¹⁰ and R^(N) are independently for each occurrence H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedcycloalkyl, optionally substituted aralkyl, optionally substitutedheteroaryl or an amino protecting group;

R^(L) is hydrogen or a ligand;

R^(P) is independently for each occurrence H, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl,optionally substituted aralkyl or optionally substituted heteroaryl;

Q is independently for each occurrence

Y₁, Y₂ and Y₃ are independently CR^(P), N, O, or S;

each of Z¹, Z² and Z³ independently comprises a R⁸ or an azide;

Z⁴ and Z⁵ are each independently for each occurrence O, S or optionallysubstituted alkyl;

n is 0-20; and

provided that at least one R⁸ or azide is present, and when R⁸ ispresent then an azide is not present in the same compound and when anazide is present then R⁸ is not present in the same compound.

In some embodiments, compounds of formula (XIX) are as shown in FIG. 12.

In one embodiment, R^(L) is selected from:

Linkers

The term “linker” means an organic moiety that connects two parts of acompound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR¹, S—S, C(O), C(O)NH, SO, SO₂, SO₂NHor a chain of atoms, such as substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl,heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl,heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R¹)₂, C(O), substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted heterocyclic; where R¹ is hydrogen, acyl, aliphatic orsubstituted aliphatic. It is further understood that linker can benon-cleavable or cleavable.

In one embodiment, the linker is C1-C30 alkyl, optionally interruptedwith at least one O, S, NR^(N), N(CO)O or combinations thereof.

In one embodiment, the linker is C1-C12 alkyl.

In one emodiment, the linker is represented by structure

—[P-Q₁-R]_(q)-T-,

wherein:

P, R and T are each independently for each occurrence absent, CO, NH, O,S, S—S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, —C(O)-(optionally substituted alkyl)-NH—, CH═N—O,

acetal, ketal,

Q₁ is independently for each occurrence absent, —(CH₂)_(n)—,—C(R¹⁰⁰)(R²⁰⁰)(CH₂)_(n)—, —(CH₂)_(n)C(R¹⁰⁰)(R²⁰⁰)—,—(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—;

R^(a) is H or an amino acid side chain;

R¹⁰⁰ and R²⁰⁰ are each independently for each occurrence H, CH₃, OH, SHor N(R^(X))₂;

R^(X) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

q is independently for each occurrence 0-20;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker has the structure—[(P-Q₁-R)_(q)—X—(P′-Q₁′-R′)_(q′)]_(q″)-T, wherein:

P, R, T, P′, R′ and T′ are each independently for each occurrenceabsent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, —C(O)-(optionally substituted alkyl)-NH—, acetal,ketal, CH═N—O,

Q₁ and Q₁′ are each independently for each occurrence absent,—(CH₂)_(n)—, —C(R¹⁰⁰)(R²⁰⁰)(CH₂)_(n)—, —(CH₂)_(n)C(R¹⁰⁰)(R²⁰⁰)—,—(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—;

X is a cleavable linker;

R^(a) is H or an amino acid side chain;

R¹⁰⁰ and R²⁰⁰ are each independently for each occurrence H, CH₃, OH, SHor N(R^(X))₂;

R^(X) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

q, q′ and q′ are each independently for each occurrence 0-20;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker comprises at least one cleavable linker.

In one embodiment, the ribose sugar of formula (I) has the structureshown in formula (I′).

wherein variable are as defined above for formula (I).

In one embodiment, the ribose sugar of formula (I) has the structureshown in formula (I″).

wherein variable are as defined above for formula (I).

Cleavable Linker

A cleavable linker is one which is sufficiently stable outside the cell,but which upon entry into a target cell is cleaved to release the twoparts the linker is holding together. In a preferred embodiment, thecleavable linker is cleaved at least 10 times or more, preferably atleast 100 times faster in the target cell or under a first referencecondition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum).

Cleavable linkers are susceptible to cleavage agents, e.g., pH, redoxpotential or the presence of degradative molecules. Generally, cleavageagents are more prevalent or found at higher levels or activities insidecells than in serum or blood. Examples of such degradative agentsinclude: redox agents which are selected for particular substrates orwhich have no substrate specificity, including, e.g., oxidative orreductive enzymes or reductive agents such as mercaptans, present incells, that can degrade a redox cleavable linker by reduction;esterases; endosomes or agents that can create an acidic environment,e.g., those that result in a pH of five or lower; enzymes that canhydrolyze or degrade an acid cleavable linker by acting as a generalacid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linker, such as a disulfide bond can be susceptible to pH.The pH of human serum is 7.4, while the average intracellular pH isslightly lower, ranging from about 7.1-7.3. Endosomes have a more acidicpH, in the range of 5.5-6.0, and lysosomes have an even more acidic pHat around 5.0. Some spacers will have a linker that is cleaved at apreferred pH, thereby releasing the iRNA agent from the carrier oligomerinside the cell, or into the desired compartment of the cell.

A spacer can include a linker that is cleavable by a particular enzyme.The type of linker incorporated into a spacer can depend on the cell tobe targeted by the iRNA agent. For example, an iRNA agent that targetsan mRNA in liver cells can be linked to the carrier oligomer through aspacer that includes an ester group. Liver cells are rich in esterases,and therefore the tether will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Cleavage of the spacerreleases the iRNA agent from the carrier oligomer, thereby potentiallyenhancing silencing activity of the iRNA agent. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Spacers that contain peptide bonds can be used when the iRNA agents aretargeting cell types rich in peptidases, such as liver cells andsynoviocytes. For example, an iRNA agent targeted to synoviocytes, suchas for the treatment of an inflammatory disease (e.g., rheumatoidarthritis), can be linked to a carrier oligomer through spacer thatcomprises a peptide bond.

In general, the suitability of a candidate cleavable linker can beevaluated by testing the ability of a degradative agent (or condition)to cleave the candidate linker. It will also be desirable to also testthe candidate cleavable linker for the ability to resist cleavage in theblood or when in contact with other non-target tissue, e.g., tissue theiRNA agent would be exposed to when administered to a subject. Thus onecan determine the relative susceptibility to cleavage between a firstand a second condition, where the first is selected to be indicative ofcleavage in a target cell and the second is selected to be indicative ofcleavage in other tissues or biological fluids, e.g., blood or serum.The evaluations can be carried out in cell free systems, in cells, incell culture, in organ or tissue culture, or in whole animals. It may beuseful to make initial evaluations in cell-free or culture conditionsand to confirm by further evaluations in whole animals. In preferredembodiments, useful candidate compounds are cleaved at least 2, 4, 10 or100 times faster in the cell (or under in vitro conditions selected tomimic intracellular conditions) as compared to blood or serum (or underin vitro conditions selected to mimic extracellular conditions).

Redox Cleavable Linkers

One class of cleavable linkers are redox cleavable linkers that arecleaved upon reduction or oxidation. An example of reductively cleavablelinker is a disulphide linker (—S—S—). To determine if a candidatecleavable linker is a suitable “reductively cleavable linker,” or forexample is suitable for use with a particular iRNA moiety and particulartargeting agent one can look to methods described herein. For example, acandidate can be evaluated by incubation with dithiothreitol (DTT), orother reducing agent using reagents know in the art, which mimic therate of cleavage which would be observed in a cell, e.g., a target cell.The candidates can also be evaluated under conditions which are selectedto mimic blood or serum conditions. In a preferred embodiment, candidatecompounds are cleaved by at most 10% in the blood. In preferredembodiments, useful candidate compounds are degraded at least 2, 4, 10or 100 times faster in the cell (or under in vitro conditions selectedto mimic intracellular conditions) as compared to blood (or under invitro conditions selected to mimic extracellular conditions). The rateof cleavage of candidate compounds can be determined using standardenzyme kinetics assays under conditions chosen to mimic intracellularmedia and compared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linkers

Phosphate-based linkers are cleaved by agents that degrade or hydrolyzethe phosphate group. An example of an agent that cleaves phosphategroups in cells are enzymes such as phosphatases in cells. Examples ofphosphate-based linkers are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—,—O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—,—O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—,—S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—.Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferredembodiment is —O—P(O)(OH)—O—. These candidates can be evaluated usingmethods analogous to those described above.

Acid Cleavable Linkers

Acid cleavable linkers are linkers that are cleaved under acidicconditions. In preferred embodiments acid cleavable linkers are cleavedin an acidic environment with a pH of about 6.5 or lower (e.g., about6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as ageneral acid. In a cell, specific low pH organelles, such as endosomesand lysosomes can provide a cleaving environment for acid cleavablelinkers. Examples of acid cleavable linkers include but are not limitedto hydrazones, esters, and esters of amino acids. Acid cleavable groupscan have the general formula —C═NN—, C(O)O, or —OC(O). A preferredembodiment is when the carbon attached to the oxygen of the ester (thealkoxy group) is an aryl group, substituted alkyl group, or tertiaryalkyl group such as dimethyl pentyl or t-butyl. These candidates can beevaluated using methods analogous to those described above.

Ester-Based Linkers

Ester-based linkers are cleaved by enzymes such as esterases andamidases in cells. Examples of ester-based cleavable linkers include butare not limited to esters of alkylene, alkenylene and alkynylene groups.Ester cleavable linkers have the general formula —C(O)O—, or —OC(O)—.These candidates can be evaluated using methods analogous to thosedescribed above.

Peptide-Based Cleaving Groups

Peptide-based linkers are cleaved by enzymes such as peptidases andproteases in cells.

Peptide-based cleavable linkers are peptide bonds formed between aminoacids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) andpolypeptides. Peptide-based cleavable groups do not include the amidegroup (—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptidecleavable linkers have the general formula —NHCHR¹C(O)NHCHR²C(O)—, whereR¹ and R² are the R groups of the two adjacent amino acids. Thesecandidates can be evaluated using methods analogous to those describedabove.

“Click” Reaction

The synthesis methods of the present invention utilize click chemistryto conjugate the ligand to the click-carrier compound. Click chemistrytechniques are described, for example, in the following references,which are incorporated herein by reference in their entirety:

-   Kolb, H. C.; Finn, M. G. and Sharpless, K. B. Angew. Chem., Int.    Ed. (2001) 40: 2004-2021.-   Kolb, H. C. and Shrapless, K. B. Drug Disc. Today (2003) 8:    112-1137.-   Rostovtsev, V. V.; Green L. G.; Fokin, V. V. and Shrapless, K. B.    Angew. Chem., Int. Ed. (2002) 41: 2596-2599.-   Tornøe, C. W.; Christensen, C. and Meldal, M. J. Org. Chem. (2002)    67: 3057-3064.-   Wang, Q. et al., J. Am. Chem. Soc. (2003) 125: 3192-3193.-   Lee, L. V. et al., J. Am. Chem. Soc. (2003) 125: 9588-9589.-   Lewis, W. G. et al., Angew. Chem., Int. Ed. (2002) 41: 1053-1057.-   Manetsch, R. et al., J. Am. Chem. Soc. (2004) 126: 12809-12818.-   Mocharla, V. P. et al., Angew. Chem., Int. Ed. (2005) 44: 116-120.

Although other click chemistry functional groups can be utilized, suchas those described in the above references, the use of cycloadditionreactions is preferred, particularly the reaction of azides with alkynylgroups. In the presence of Cu(I) salts, terminal alkynes and azidesundergo 1,3-dipolar cycloaddition forming 1,4-disubstituted1,2,3-triazoles. In the presence of Ru(II) salts (e.g. Cu*RuCl(PPh₃)₂),terminal alkynes and azides under go 1,3-dipolar cycloaddition forming1,5-disubstituted 1,2,3-triazoles (Folkin, V. V. et al., Org. Lett.(2005) 127: 15998-15999). Alternatively, a 1,5-disubstituted1,2,3-triazole can be formed using azide and alkynyl reagents (Kraniski,A.; Fokin, V. V. and Sharpless, K. B. Org. Lett. (2004) 6: 1237-1240.Hetero-Diels-Alder reactions or 1,3-dipolar cycloaddition reaction couldalso be used (see for example Padwa, A. 1,3-Dipolar CycloadditionChemistry: Volume 1, John Wiley, New York, (1984) 1-176; Jorgensen, K.A. Angew. Chem., Int. Ed. (2000) 39: 3558-3588 and Tietze, L. F. andKettschau, G. Top. Curr. Chem. (1997) 189: 1-120)

The choice of azides and alkynes as coupling partners is particularlyadvantageous as they are essentially non-reactive towards each other (inthe absence of copper) and are extremely tolerant of other functionalgroups and reaction conditions. This chemical compatibility helps ensurethat many different types of azides and alkynes may be coupled with eachother with a minimal amount of side reactions.

The required copper(I) species are added directly as cuprous salts, forexample CuI, CuOTf.C₆H₆ or [Cu(CH₃CN)₄][PF₆], usually with stabilizingligands (see for example Tornøe, C. W.; Christensen, C. and Meldal, M.J. Org. Chem. (2002) 67: 3057-3064; Chan, T. R. et al., Org. Lett.(2004) 6: 2853-2855; Lewis, W. G. et al., J. Am. Chem. Soc. (2004) 126:9152-9153; Mantovani, G. et al., Chem. Comm. (2005) 2089-2091;Diez-Gonzalez, S. et al., Chem. Eur. J. (2006) 12: 7558-7564 andCandelon, N. et al., Chem. Comm. (2008) 741-743), or more oftengenerated from copper (II) salts with reducing agents (Rostovtsev, V. V.et al., Angew. Chem. (2002) 114: 2708-2711 and Angew. Chem., Int. Ed.(2002) 41: 2596-2599). Metallic copper (for example see Himo, F. et al.,J. Am. Chem. Soc. (2005) 127: 210-216) or clusters (for example seePachon, L. D. et al., Adv. Synth. Catal. (2005) 347: 811-815 andMolteni, G. et al., New J. Chem (2006) 30: 1137-1139) can also beemployed. Chassaing et al., recently reported copper(I) zeolites ascatalysts for the azide-alkyne cycloaddition (Chem. Eur. J. (2008) 14:6713-6721). As copper(I) salts are prone to redox process, nitrogen- orphosphorous-based ligands must be added to protect and stabilize theactive copper catalyst during the cycloaddition reaction.

The reaction is extremely straightforward. The azide and alkyne areusually mixed together in water and a co-solvent such as tert-butanol,THF, DMSO, toluene or DMF. The water/co-solvent are usually in a 1:1 to1:9 ratio. The reactions are usually run overnight although mild heatingshortens reaction times (Sharpless, W. D.; Wu, P.; Hansen, T. V.; andLi, J. G. J. Chem. Ed. (2005) 82: 1833). Aqueous systems can also usecopper(I) species directly such that a reducing agent is not needed. Thereactions conditions then usually require acetonitrile as a co-solvent(although not essential (Chan, T. R.; Hilgraf, R.; Shrapless, K. B. andFokin, V. V. Org. Lett. (2004) 6: 2853)) and a nitrogen base, such astriethylamine, 2,6-lutidine, pyridine and diisopropylamine. In this casecopper(I) species is supplied as CuI, CuOTf.C₆H₆, or[Cu(CH₃CN)₄][PF₆](Rostoctsev, V. V.; Green L. G.; Fokin, V. V. andShrapless, K. B. Angew. Chem., Int. Ed. (2002) 41: 2596-2599).

Although the water-based methods are attractive for many applications,solvent based azide-alkyne cycloaddition methods have found utility insituations when solubility and/or other problems arise, for example see:

-   Malkoch, M. et al., Macromolecules (2005) 38: 3663.-   Gujadhur, R.; Venkataraman, D. and J. T. Kintigh. J. T. Tet.    Lett. (2001) 42: 4791.-   Laurent, B. A. and Grayson, S. M. J. Am. Chem. Soc. (2006) 128:    4238.-   Opsteen, J. A.; van Hest, J. C. M. Chem. Commun. (2005) 57.-   Tsarevsky, N. V.; Sumerlin, B. S. and Matyjaszewski, K.    Macromolecules (2005) 38: 3558.-   Johnson, J. A. et al., J. Am. Chem. Soc. (2006) 128: 6564.-   Sumerlin, B. S. et al., Macromolecules (2005) 38: 7540.-   Gao, H. F. and Matyjaszewski, K. Macromolecules (2006) 39: 4960.-   Gao, H. et al, Macromolecules (2005) 38: 8979.-   Vogt, A. P. and Sumerlin, B. S. Macromolecules (2006) 39: 5286.-   Lutz, J. F.; Borner, H. G. and Weichenhan, K. Macromol. Rapid    Comm. (2005) 26: 514.-   Mantovani, G.; Ladmiral, V.; Tao, L. and Haddleton, D. M. Chem.    Comm. (2005) 2089.

The click reaction may be performed thermally. In one aspect, the clickreaction is performed at slightly elevated temperatures between 25° C.and 100° C. In one aspect, the reaction may be performed between 25° C.and 75° C., or between 25° C. and 65° C., or between 25° C. and 50° C.In one embodiment, the reaction is performed at room temperature. Inanother aspect, the click reaction may also be performed using amicrowave oven. The microwave assisted click reaction may be carried outin the presence or absence of copper.

In one aspect, the invention provides a method for coupling aclick-carrier compound to a ligand through a click reaction. In apreferred embodiment, the click reaction is a cycloaddition reaction ofazide with alkynyl group and catalyzed by copper. In one embodiment theequal molar amount of alkyne and azide are mixed together in DCM/MeOH(10:1 to 1:1 ratio v/v) and 0.05-0.5 mol % each of [Cu(CH₃CN)₄][PF₆] andcopper are added the reaction. In one embodiment DCM/MeOH ratio is 5:1to 1:1. In a preferred embodiment, DCM/MeOH ratio is 4:1. In oneembodiment, equal molar amounts of [Cu(CH₃CN)₄][PF₆] and copper areadded. In a preferred embodiment, 0.05-0.25 mol % each of[Cu(CH₃CN)₄][PF₆] and copper are added to the reaction. In a morepreferred embodiment, 0.05 mol %, 0.1 mol %, 0.15 mol %, 0.2 mol % or0.25 mol % each of [Cu(CH₃CN)₄][PF₆] and copper are added to thereaction.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the oligomeric compounds of theinvention: i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

Ligands

A wide variety of entities can be coupled to the oligonucleotide, e.g.the iRNA agent, using the “click” reaction. Preferred entities can becoupled to the oligonucleotide at various places, for example, 3′-end,5′-end, and/or at internal positions.

In preferred embodiments, the ligand is attached to the iRNA agent viaan intervening linker. The ligand may be present on a compound when saidcompound is incorporated into the growing strand. In some embodiments,the ligand may be incorporated via coupling to a “precursor” compoundafter said “precursor” compound has been incorporated into the growingstrand. For example, a compound having, e.g., an azide terminated linker(i.e., having no associated ligand), e.g., -linker-N₃ may beincorporated into a growing sense or antisense strand. In a subsequentoperation, i.e., after incorporation of the precursor compound into thestrand, a ligand having an alkyne, e.g. terminal acetylene, e.g.ligand-C≡CH, can subsequently be attached to the precursor compound bythe “click” reaction. Alternatively, the compound linker comprises analkyne, e.g. terminal acetylene; and the ligand comprises an azidefunctionality for the “click” reaction to take place. The azide oralkyne functionalities can be incorporated into the ligand by methodsknown in the art. For example, moieties carrying azide or alkynefunctionalities can be linked to the ligand or a functional group on theligand can be transformed into an azide or alkyne. In one embodiment,the conjugation of the ligand to the precursor compound takes placewhile the oligonucleotide is still attached to the solid support. In oneembodiment, the precursor carrying oligonucleotide is first deprotectedbut not purified before the ligand conjugation takes place. In oneembodiment, the precursor compound carrying oligonucleotide is firstdeprotected and purified before the ligand conjugation takes place. Incertain embodiments, the “click” reaction is carried out undermicrowave.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Preferred ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In certain embodiments, the endosomolytic ligand assumesits active conformation at endosomal pH. The “active” conformation isthat conformation in which the endosomolytic ligand promotes lysis ofthe endosome and/or transport of the composition of the invention, orits components, from the endosome to the cytoplasm of the cell.Exemplary endosomolytic ligands include the GALA peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al.,J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments,the endosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched. Exemplary primary sequences of peptide based endosomolyticligands are shown in table 1.

TABLE 1 List of peptides with endosomolytic activity. SEQ ID NameSequence (N to C) Ref. NO: GALA AALEALAEALEALAEALEALAEAAAAGGC 1  1 EALAAALAEALAEALAEALAEALAEALAAAAGGC 2  2 ALEALAEALEALAEA 3  3 INF-7GLFEAIEGFIENGWEGMIWDYG 4  4 Inf  GLFGAIAGFIENGWEGMIDGWYG 5  5 HA-2diINF-7 GLF EAI EGFI ENGW EGMI DGWYGC 5  6 GLF EAI EGFI ENGW EGMI DGWYGC 6 diINF3 GLF EAI EGFI ENGW EGMI DGGC 6  7 GLF EAI EGFI ENGW EGMI DGGC 7 GLF GLFGALAEALAEALAEHLAEALAEALEALAAGG 6  8 SC GALA-GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC 6  9 INF3 INF-5GLF EAI EGFI ENGW EGnI DG K 4 10 GLF EAI EGFI ENGW EGnI DG 11 n,norleucine References 1 Subbarao et al. (1987) Biochemistry 26:2964-2972. 2 Vogel, et al. (1996)J. Am. Chem. Soc. 118: 1581-1586 3Turk, et al. (2002) Biochim. Biophys. Acta 1559: 56-68. 4 Plank, et al.(1994) J. Biol. Chem. 269: 12918-12924. 5 Mastrobattista, et al. (2002)J. Biol. Chem. 277: 27135-43. 6 Oberhauser, et al. (1995) Deliv.Strategies Antisense Oligonucleotide Ther. 247-66.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of compounds described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 2 shows some examples of targetingligands and their associated receptors.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

TABLE 2 Liver targeting Ligands and their associated receptors LiverCells Ligand Receptor 1) Parenchymal Galactose ASGP-R Cell (PC)(Asiologlycoprotein (Hepatocytes) receptor) Gal NAc (n-acetyl- ASPG-R(GalNAc galactosamine) Receptor) Lactose Asialofetuin ASPG-r 2)Sinusoidal Hyaluronan Hyaluronan receptor Endothelial Cell ProcollagenProcollagen receptor (SEC) Negatively charged molecules Scavengerreceptors Mannose Mannose receptors N-acetyl Glucosamine Scavengerreceptors Immunoglobulins Fc Receptor LPS CD14 Receptor Insulin Receptormediated transcytosis Transferrin Receptor mediated transcytosisAlbumins Non-specific Sugar-Albumin conjugates Mannose-6-phosphateMannose-6-phosphate receptor 3) Kupffer Cell Mannose Mannose receptors(KC) Fucose Fucose receptors Albumins Non-specific Mannose-albuminconjugates

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver.

Other molecules that can bind HSA can also be used as ligands. Forexample, neproxin or aspirin can be used. A lipid or lipid-based ligandcan (a) increase resistance to degradation of the conjugate, (b)increase targeting or transport into a target cell or cell membrane,and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells.

Exemplary vitamins include vitamin A, E, and K. Other exemplary vitaminsinclude are B vitamin, e.g., folic acid, B12, riboflavin, biotin,pyridoxal or other vitamins or nutrients taken up by cancer cells. Alsoincluded are HAS, low density lipoprotein (LDL) and high-densitylipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 3, forexample).

TABLE 3 Exemplary Cell Permeation Peptides Cell Permea- SEQ tion IDPeptide Amino acid Sequence NO: Reference Pene- RQIKIWFQNRRMKWKK 12Derossi et  tratin al., J.  Biol. Chem. 269:  10444, 1994 Tat GRKKRRQRRRPPQ 13 Vives et   fragment al., J.  (48-60) Biol.  Chem., 272:16010, 1997 Signal GALFLGWLGAAGSTMGAWSQP 14 Chaloin et  Sequence- KKKRKVal., based   Biochem.  peptide Biophys. Res.  Commun., 243: 601,  1998PVEC LLIILRRRIRKQAHAHSK 15 Elmquist   et al.,  Exp. Cell Res., 269: 237, 2001 Trans- GWTLNSAGYLLKINLKALAALA 16 Pooga et   portan KKILal., FASEB  J., 12: 67, 1998 Amphiphi- KLALKLALKALKAALKLA 17 Oehlke et  lic al., Mol.  model Ther., 2:  peptide 339, 2000 Arg₉ RRRRRRRRR 18Mitchell   et al., J. Pept. Res., 56:  318, 2000 Bacterial  KFFKFFKFFK19 cell wall permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQRI 20 KDFLRNLVPRTESCecropin  SWLSKTAKKLENSAKKRISEGIAI 21 P1 AIQGGPR α-defensinACYCRIPACIAGERRYGTCIYQGR 22 LWAFCC b-defensin DHYNCVSSGGQCLYSACPIFTKIQ23 GTCYRGKAKCCK ) Bactenecin RKCRIVVIRVCR 24 PR-39RRRPRPPYLPRPRPPPFFPPRLPPR 25 IPPGFPPRFPPRFPGKR-NH₂ Indolici-ILPWKWPWWPWRR-NH₂ 26 din

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 27). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 28)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 13)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 29))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered toan iRNA agent via an incorporated compound unit is a cell targetingpeptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGDmimic. A peptide moiety can range in length from about 5 amino acids toabout 40 amino acids. The peptide moieties can have a structuralmodification, such as to increase stability or direct conformationalproperties. Any of the structural modifications described below can beutilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing α_(V)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an I_(v)θ₃ integrin.Thus, one could use RGD peptides, cyclic peptides containing RGD, RGDpeptides that include D-amino acids, as well as synthetic RGD mimics. Inaddition to RGD, one can use other moieties that target the I_(v)-θ₃integrin ligand. Generally, such ligands can be used to controlproliferating cells and angiogeneis. Preferred conjugates of this typeligands that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancergene described herein.

TABLE 4 Azide modified peptides (SEQ ID NOS 30, 30,31, 31, 18, and 18, respectively, in order of appearance). NB12675N₃-(CH₂)₅-CO-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-NH₂ NB12707N₃-(CH₂)₁₅-CO-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-NH₂ NB12676cyclo-[Phe-Arg-Gly-Asp-Lys(N₃-(CH₂)₅- COOH)] NB12708cyclo-[Phe-Arg-Gly-Asp-Lys(N₃-(CH₂)₁₅- COOH)] NB12709N₃-(CH2)₅-CO-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-NH₂ NB12710N₃-(CH2)₁₅-CO-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-NH₂

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an iRNA agent and/orthe carrier oligomer can be an amphipathic α-helical peptide. Exemplaryamphipathic α-helical peptides include, but are not limited to,cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide(BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfishintestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2,dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides,esculentinis-1, and caerins. A number of factors will preferably beconsidered to maintain the integrity of helix stability. For example, amaximum number of helix stabilization residues will be utilized (e.g.,leu, ala, or lys), and a minimum number helix destabilization residueswill be utilized (e.g., proline, or cyclic compoundic units. The cappingresidue will be considered (for example Gly is an exemplary N-cappingresidue and/or C-terminal amidation can be used to provide an extraH-bond to stabilize the helix. Formation of salt bridges betweenresidues with opposite charges, separated by i±3, or i±4 positions canprovide stability. For example, cationic residues such as lysine,arginine, homo-arginine, ornithine or histidine can form salt bridgeswith the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, GalNAc₃, galactose,mannose, mannose-6P, clusters of sugars such as GalNAc cluster, mannosecluster, galactose cluster, or an apatamer. A cluster is a combinationof two or more sugar units. The targeting ligands also include integrinreceptor ligands, Chemokine receptor ligands, transferrin, biotin,serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDLand HDL ligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycaboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Exemplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligands amenable to the invention are described in copendingapplications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser.No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filedAug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser.No. 11/944,227 filed Nov. 21, 2007, which are incorporated by referencein their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

The compound comprising the ligand, e.g. the click-carrier compound, canbe present in any position of an oligonucleotide, e.g. an iRNA agent. Insome embodiments, click-carrier compound can be present at the terminussuch as a 5′ or 3′ terminal of the iRNA agent. Click-carrier compoundscan also present at an internal position of the iRNA agent. Fordouble-stranded iRNA agents, click-carrier compounds can be incorporatedinto one or both strands. In some embodiments, the sense strand of thedouble-stranded iRNA agent comprises the click-carrier compound. Inother embodiments, the antisense strand of the double-stranded iRNAagent comprises the click-carrier compound.

In some embodiments, ligands can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

There are numerous methods for preparing conjugates of oligomericcompounds. Generally, an oligomeric compound is attached to a conjugatemoiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl,aldehyde, and the like) on the oligomeric compound with a reactive groupon the conjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic.

For example, an electrophilic group can be a carbonyl-containingfunctionality and a nucleophilic group can be an amine or thiol. Methodsfor conjugation of nucleic acids and related oligomeric compounds withand without linkers are well described in the literature such as, forexample, in Manoharan in Antisense Research and Applications, Crooke andLeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation ofoligonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,149,782; 5,214,136;5,245,022; 5,254, 469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662;5,688,941; 5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434;6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631;6,559,279; each of which is herein incorporated by reference.

Oligonucleotide

The term “oligonucleotide” as used herein refers to an unmodified RNA,modified RNA, or nucleoside surrogate, all of which are defined herein.Although the modifications are described in context of an iRNA agent, itis understood that these modifications are also applicable to otheroligonucleotides of the invention such as antisense, antagomir, aptamer,ribozyme and decoy oligonucleotides. While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Preferred examples include those which have a 2′ sugarmodification, a modification in a single strand overhang, preferably a3′ single strand overhang, or, particularly if single stranded, a 5′modification which includes one or more phosphate groups or one or moreanalogs of a phosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, down regulate the expression ofa target gene, preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent caninclude a single strand or can include more than one strands, e.g., itcan be a double stranded iRNA agent. If the iRNA agent is a singlestrand it is particularly preferred that it include a 5′ modificationwhich includes one or more phosphate groups or one or more analogs of aphosphate group. If the iRNA agent is double stranded the doublestranded region can include more than two or more strands, e.g, twostrands, e.g. three strands, in the double stranded region.

The iRNA agent should include a region of sufficient homology to thetarget gene, and be of sufficient length in terms of nucleotides, suchthat the iRNA agent, or a fragment thereof, can mediate down regulationof the target gene. (For ease of exposition the term nucleotide orribonucleotide is sometimes used herein in reference to one or morecompoundic subunits of an RNA agent. It will be understood herein thatthe usage of the term “ribonucleotide” or “nucleotide”, herein can, inthe case of a modified RNA or nucleotide surrogate, also refer to amodified nucleotide, or surrogate replacement moiety at one or morepositions.) Thus, the iRNA agent is or includes a region which is atleast partially, and in some embodiments fully, complementary to thetarget RNA. It is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence must besufficient to enable the iRNA agent, or a cleavage product thereof, todirect sequence specific silencing, e.g., by RNAi cleavage of the targetRNA, e.g., mRNA.

An iRNA agent will often be modified or include nucleoside surrogates inaddition to the click-carrier compound. Single stranded regions of aniRNA agent will often be modified or include nucleoside surrogates,e.g., the unpaired region or regions of a hairpin structure, e.g., aregion which links two complementary regions, can have modifications ornucleoside surrogates. Modification to stabilize one or more 3′- or5′-terminus of an iRNA agent, e.g., against exonucleases, or to favorthe antisense sRNA agent to enter into RISC are also favored.Modifications can include C3 (or C6, C7, C12) amino linkers, thiollinkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12,abasic, triethylene glycol, hexaethylene glycol), special biotin orfluorescein reagents that come as phosphoramidites and that have anotherDMT-protected hydroxyl group, allowing multiple couplings during RNAsynthesis.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. In preferred embodiments singlestrand iRNA agents are 5′-phosphorylated or include a phosphoryl analogat the 5′-terminus. 5′-phosphate modifications include those which arecompatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). (These modifications can also be used with theantisense strand of a multi-strand iRNA agent.)

A “multi-strand iRNA agent” as used herein, is an iRNA agent whichcomprises two or more strands, for example a double-stranded iRNA agent.The strands form duplexed regions and may include a hairpin, pan-handlestructure, loop or bulges. At least one strand of the iRNA agent ispreferably antisense with regard to the target molecule.

It may be desirable to modify only one, only two or all strands of amulti-strand iRNA agent. In some cases they will have the samemodification or the same class of modification but in other cases thedifferent strand will have different modifications, e.g., in some casesit is desirable to modify only one strand. It may be desirable to modifyonly some strands, e.g., to inactivate them, e.g., strands can bemodified in order to inactivate them and prevent formation of an activeiRNA/protein or RISC. This can be accomplished by a modification whichprevents 5′-phosphorylation of the strands, e.g., by modification with a5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATP requirementsand small interfering RNA structure in the RNA interference pathway.Cell 107, 309-321.) Other modifications which prevent phosphorylationcan also be used, e.g., simply substituting the 5′-OH by H rather thanO-Me. Alternatively, a large bulky group may be added to the5′-phosphate turning it into a phosphodiester linkage, though this maybe less desirable as phosphodiesterases can cleave such a linkage andrelease a functional iRNA 5′-end. Antisense strand modifications include5′-phosphorylation as well as any of the other 5′ modificationsdiscussed herein, particularly the 5′ modifications discussed above inthe section on single stranded iRNA molecules.

In some cases, the different strands will include differentmodifications. Multiple different modifications can be included on eachof the strands. The modifications on a given strand may differ from eachother, and may also differ from the various modifications on otherstrands. For example, one strand may have a modification, e.g., amodification described herein, and a different strand may have adifferent modification, e.g., a different modification described herein.In other cases, one strand may have two or more different modifications,and the another strand may include a modification that differs from theat least two modifications on the other strand.

It is preferred that the strands be chosen such that the iRNA agentincludes a single strand or unpaired region at one or both ends of themolecule. Thus, an iRNA agent contains two or more strands, preferablepaired to contain an overhang, e.g., one or two 5′ or 3′ overhangs butpreferably a 3′ overhang of 2-3 nucleotides. Most embodiments will havea 3′ overhang. Preferred iRNA agents will have single-strandedoverhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3nucleotides in length at each end. The overhangs can be the result ofone strand being longer than the other, or the result of two strands ofthe same length being staggered.

Preferred lengths for the duplexed regions between the strands arebetween 6 and 30 nucleotides in length. The preferred duplexed regionsare between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23nucleotides in length. Other preferred duplexed regions are between 6and 20 nucleotides, most preferably 6, 7, 8, 9, 10, 11 and 12nucleotides in length. In multi-strand iRNA agents different duplexesformed may have different lengths, e.g. duplexed region formed betweenstrand A and B may have a different length than duplexed region formedbetween strand A and C.

In iRNA agents comprising more than two strands duplexed agents canresemble in length and structure the natural Dicer processed productsfrom long dsRNAs. Embodiments in which the two or more strands of theiRNA agent are linked, e.g., covalently linked are also included.Hairpins or other single strand structures which provide the requireddouble stranded region, and preferably a 3′ overhang are also within theinvention.

As nucleic acids are polymers of subunits or compounds, many of themodifications described below occur at a position which is repeatedwithin a nucleic acid, e.g., a modification of a base, or a phosphatemoiety, or the non-bridging oxygen of a phosphate moiety. In some casesthe modification will occur at all of the subject positions in thenucleic acid but in many, and in fact in most cases it will not. By wayof example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in the internal unpaired region, may only occurin a terminal regions, e.g. at a position on a terminal nucleotide or inthe last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification mayoccur in a double strand region, a single strand region, or in both. Amodification may occur only in the double strand region of an RNA agentor may only occur in a single strand region of an RNA agent. E.g., aphosphorothioate modification at a non-bridging oxygen position may onlyoccur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. The 5′ end or ends can bephosphorylated.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular bases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Specific modifications are discussed in more detail below. Although, themodifications herein are described in context of an iRNA agent, thesemodifications are also amenable in modifying the carrieroligonucleotides of the invention.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-bridging oxygen atoms. However, thephosphate group can be modified by replacing one of the oxygens with adifferent substituent. One result of this modification to RNA phosphatebackbones can be increased resistance of the oligoribonucleotide tonucleolytic breakdown. Thus while not wishing to be bound by theory, itcan be desirable in some embodiments to introduce alterations whichresult in either an uncharged linker or a charged linker withunsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-bridging oxygensreplaced by sulfur. The phosphorus center in the phosphorodithioates isachiral which precludes the formation of oligoribonucleotidesdiastereomers. Diastereomer formation can result in a preparation inwhich the individual diastereomers exhibit varying resistance tonucleases. Further, the hybridization affinity of RNA containing chiralphosphate groups can be lower relative to the corresponding unmodifiedRNA species. Thus, while not wishing to be bound by theory,modifications to both non-bridging oxygens, which eliminate the chiralcenter, e.g. phosphorodithioate formation, may be desirable in that theycannot produce diastereomer mixtures. Thus, the non-bridging oxygens canbe independently any one of S, Se, B, C, H, N, or OR (R is alkyl oraryl). Replacement of the non-bridging oxygens with sulfur is preferred.

The phosphate linker can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur atthe either linking oxygen or at both the linking oxygens. When thebridging oxygen is the 3′-oxygen of a nucleoside, replacement withcarbon is preferred. When the bridging oxygen is the 5′-oxygen of anucleoside, replacement with nitrogen is preferred.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors. While not wishing to be bound by theory, it is believed thatsince the charged phosphodiester group is the reaction center innucleolytic degradation, its replacement with neutral structural mimicsshould impart enhanced nuclease stability. Again, while not wishing tobe bound by theory, it can be desirable, in some embodiment, tointroduce alterations in which the charged phosphate group is replacedby a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.Preferred replacements include the methylenecarbonylamino andmethylenemethylimino groups.

Modified phosphate linkages where at least one of the oxygens linked tothe phosphate has been replaced or the phosphate group has been replacedby a non-phosphorous group, are also referred to as “non phosphodiesterbackbone linkage.”

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. While not wishing to be bound bytheory, it is believed that the absence of a repetitively chargedbackbone diminishes binding to proteins that recognize polyanions (e.g.nucleases). Again, while not wishing to be bound by theory, it can bedesirable in some embodiment, to introduce alterations in which thebases are tethered by a neutral surrogate backbone. Examples include themorphilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA)nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Sugar Modifications

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a2′-alkoxide ion. The 2′-alkoxide can catalyze degradation byintramolecular nucleophilic attack on the linker phosphorus atom. Again,while not wishing to be bound by theory, it can be desirable to someembodiments to introduce alterations in which alkoxide formation at the2′ position is not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or sugar), cyano; mercapto; alkyl-thioalkyl; thioalkoxy; andalkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionallysubstituted with e.g., an amino functionality. Preferred substitutentsare 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining e.g., arabinose, as the sugar.

Modified RNAs can also include “abasic” sugars, which lack a nucleobaseat C-1′. These abasic sugars can also be further contain modificationsat one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The modification can also entail the wholesale replacement of a ribosestructure with another entity at one or more sites in the iRNA agent.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotinand fluorescein reagents. When a spacer/phosphate-functional molecularentity-spacer/phosphate array is interposed between two strands of iRNAagents, this array can substitute for a hairpin RNA loop in ahairpin-type RNA agent. The 3′ end can be an —OH group. While notwishing to be bound by theory, it is believed that conjugation ofcertain moieties can improve transport, hybridization, and specificityproperties. Again, while not wishing to be bound by theory, it may bedesirable to introduce terminal alterations that improve nucleaseresistance. Other examples of terminal modifications include dyes,intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation. Terminal modifications useful for modulatingactivity include modification of the 5′ end with phosphate or phosphateanalogs. E.g., in preferred embodiments iRNA agents, especiallyantisense strands, are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Nucleobases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the abovebases, e.g., “unusual bases”, “modified bases”, “non-natural bases” and“universal bases” described herein, can be employed. Examples includewithout limitation 2-aminoadenine, 6-methyl and other alkyl derivativesof adenine and guanine, 2-propyl and other alkyl derivatives of adenineand guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, butthey can be useful for other reasons, e.g., some, e.g.,2,6-diaminopurine and 2 amino purine, are fluorescent. Modified basescan reduce target specificity. This should be taken into considerationin the design of iRNA agents.

Cationic Groups

Modifications can also include attachment of one or more cationic groupsto the sugar, base, and/or the phosphorus atom of a phosphate ormodified phosphate backbone moiety. A cationic group can be attached toany atom capable of substitution on a natural, unusual or universalbase. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Exemplary Modifications and Placement within an iRNA Agent

Some modifications may preferably be included on an iRNA agent at aparticular location, e.g., at an internal position of a strand, or onthe 5′ or 3′ end of a strand of an iRNA agent. A preferred location of amodification on an iRNA agent, may confer preferred properties on theagent. For example, preferred locations of particular modifications mayconfer optimum gene silencing properties, or increased resistance toendonuclease or exonuclease activity. A modification described hereinand below may be the sole modification, or the sole type of modificationincluded on multiple ribonucleotides, or a modification can be combinedwith one or more other modifications described herein and below. Forexample, a modification on one strand of a multi-strand iRNA agent canbe different than a modification on another strand of the multi-strandiRNA agent. Similarly, two different modifications on one strand candiffer from a modification on a different strand of the iRNA agent.Other additional unique modifications, without limitation, can beincorporates into strands of the iRNA agent.

An iRNA agent may include a backbone modification to any nucleotide onan iRNA strand. For example, an iRNA agent may include aphosphorothioate linkage or P-alkyl modification in the linkages betweenone or more nucleotides of an iRNA agent. The nucleotides can beterminal nucleotides, e.g., nucleotides at the last position of a senseor antisense strand, or internal nucleotides.

An iRNA agent can include a sugar modification, e.g., a 2′ or 3′ sugarmodification. Exemplary sugar modifications include, for example, a2′-O-methylated nucleotide, a 2′-deoxy nucleotide, (e.g., a2′-deoxyfluoro nucleotide), a 2′-O-methoxyethyl nucleotide, a 2′-O-NMA,a 2′-DMAEOE, a 2′-aminopropyl, 2′-hydroxy, or a 2′-ara-fluoro or alocked nucleic acid (LNA), extended nucleic acid (ENA), hexose nucleicacid (HNA), or cyclohexene nucleic acid (CeNA). A 2′ modification ispreferably 2′-OMe, and more preferably, 2′-deoxyfluoro. When themodification is 2′-OMe, the modification is preferably on the sensestrands. When the modification is a 2′-fluoro, and the modification maybe on any strand of the iRNA agent. A 2′-ara-fluoro modification willpreferably be on the sense strands of the iRNA agent. An iRNA agent mayinclude a 3′ sugar modification, e.g., a 3′-OMe modification. Preferablya 3′-OMe modification is on the sense strand of the iRNA agent.

An iRNA agent may include a 5′-methyl-pyrimidine (e.g., a5′-methyl-uridine modification or a 5′-methyl-cytodine) modification.

The modifications described herein can be combined onto a single iRNAagent. For example, an iRNA agent may have a phosphorothioate linkageand a 2′ sugar modification, e.g., a 2′-OMe or 2′-F modification. Inanother example, an iRNA agent may include at least one 5-Me-pyrimidineand a 2′-sugar modification, e.g., a 2′-F or 2′-OMe modification.

An iRNA agent may include a nucleobase modification, such as a cationicmodification, such as a 3′-abasic cationic modification. The cationicmodification can be e.g., an alkylamino-dT (e.g., a C6 amino-dT), anallylamino conjugate, a pyrrolidine conjugate, a pthalamido, aporphyrin, or a hydroxyprolinol conjugate, on one or more of theterminal nucleotides of the iRNA agent. When an alkylamino-dT conjugateis attached to the terminal nucleotide of an iRNA agent, the conjugateis preferably attached to the 3′ end of the sense or antisense strand ofan iRNA agent. When a pyrrolidine linker is attached to the terminalnucleotide of an iRNA agent, the linker is preferably attached to the3′- or 5′-end of the sense strand, or the 3′-end of the antisensestrand. When a pyrrolidine linker is attached to the terminal nucleotideof an iRNA agent, the linker is preferably on the 3′- or 5′-end of thesense strand, and not on the 5′-end of the antisense strand.

An iRNA agent may include at least one conjugate, such as a lipophile, aterpene, a protein binding agent, a vitamin, a carbohydrate, or apeptide. For example, the conjugate can be naproxen, nitroindole (oranother conjugate that contributes to stacking interactions), folate,ibuprofen, or a C5 pyrimidine linker. The conjugate can also be aglyceride lipid conjugate (e.g., a dialkyl glyceride derivatives),vitamin E conjugate, or a thio-cholesterol. In generally, and exceptwhere noted to the contrary below, when a conjugate is on the terminalnucleotide of a sense or antisense strand, the conjugate is preferablyon the 5′ or 3′ end of the sense strand or on the 5′ end of theantisense strand, and preferably the conjugate is not on the 3′ end ofthe antisense strand.

When the conjugate is naproxen, and the conjugate is on the terminalnucleotide of a sense or antisense strand, the conjugate is preferablyon the 5′ or 3′ end of the sense or antisense strands. When theconjugate is cholesterol, and the conjugate is on the terminalnucleotide of a sense or antisense strand, the cholesterol conjugate ispreferably on the 5′ or 3′ end of the sense strand and preferably notpresent on the antisense strand. Cholesterol may be conjugated to theiRNA agent by a pyrrolidine linker, serinol linker, hydroxyprolinollinker, or disulfide linkage. A dU-cholesterol conjugate may also beconjugated to the iRNA agent by a disulfide linkage. When the conjugateis cholanic acid, and the conjugate is on the terminal nucleotide of asense or antisense strand, the cholanic acid is preferably attached tothe 5′ or 3′ end of the sense strand, or the 3′ end of the antisensestrand. In one embodiment, the cholanic acid is attached to the 3′ endof the sense strand and the 3′ end of the antisense strand.

One or more nucleotides of an iRNA agent may have a 2′-5′ linkage.Preferably, the 2′-5′ linkage is on the sense strand. When the 2′-5′linkage is on the terminal nucleotide of an iRNA agent, the 2′-5′linkage occurs on the 5′ end of the sense strand.

The iRNA agent may include an L-sugar, preferably on the sense strand,and not on the antisense strand.

The iRNA agent may include a methylphosphonate modification. When themethylphosphonate is on the terminal nucleotide of an iRNA agent, themethylphosphonate is at the 3′ end of the sense or antisense strands ofthe iRNA agent.

An iRNA agent may be modified by replacing one or more ribonucleotideswith deoxyribonucleotides. Preferably, adjacent deoxyribonucleotides arejoined by phosphorothioate linkages, and the iRNA agent does not includemore than four consecutive deoxyribonucleotides on the sense or theantisense strands.

An iRNA agent may include a difluorotoluyl (DFT) modification, e.g.,2,4-difluorotoluyl uracil, or a guanidine to inosine substitution.

The iRNA agent may include at least one 5′-uridine-adenine-3′ (5′-UA-3′)dinucleotide wherein the uridine is a 2′-modified nucleotide, or aterminal 5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide, or a terminal5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide, or a terminal 5′-uridine-uridine-3′(5′-UU-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide, or a terminal 5′-cytidine-cytidine-3′ (5′-CC-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide, or aterminal 5′-cytidine-uridine-3′ (5′-CU-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide, or a terminal5′-uridine-cytidine-3′ (5′-UC-3′) dinucleotide, wherein the 5′-uridineis a 2′-modified nucleotide. The chemically modified nucleotide in theiRNA agent may be a 2′-O-methylated nucleotide. In some embodiments, themodified nucleotide can be a 2′-deoxy nucleotide, a 2′-deoxyfluoronucleotide, a 2′-O-methoxyethyl nucleotide, a 2′-O-NMA, a 2′-DMAEOE, a2′-aminopropyl, 2′-hydroxy, or a 2′-ara-fluoro, or a locked nucleic acid(LNA), extended nucleic acid (ENA), hexose nucleic acid (HNA), orcyclohexene nucleic acid (CeNA). The iRNA agents including thesemodifications are particularly stabilized against exonuclease activity,when the modified dinucleotide occurs on a terminal end of the sense orantisense strand of an iRNA agent, and are otherwise particularlystabilized against endonuclease activity.

An iRNA agent may have a single overhang, e.g., one end of the iRNAagent has a 3′ or 5′ overhang and the other end of the iRNA agent is ablunt end, or the iRNA agent may have a double overhang, e.g., both endsof the iRNA agent have a 3′ or 5′ overhang, such as a dinucleotideoverhang. In another alternative, both ends of the iRNA agent may haveblunt ends. The unpaired nucleotides may have at least onephosphorothioate dinucleotide linkage, and at least one of the unpairednucleotides may be chemically modified in the 2′-position. Thedoublestrand region of the iRNA agent may include phosphorothioatedinucleotide linkages on one or both of the sense and antisense strands.Various strands of the multi-strand iRNA agent may be connected with alinker, e.g., a chemical linker such as hexaethylene glycol linker, apoly-(oxyphosphinico-oxy-1,3-propandiol) linker, an allyl linker, or apolyethylene glycol linker.

Nuclease Resistant Compounds

An iRNA agent can include compounds which have been modified so as toinhibit degradation, e.g., by nucleases, e.g., endonucleases orexonucleases, found in the body of a subject. These compounds arereferred to herein as NRMs, or nuclease resistance promoting compoundsor modifications. In many cases these modifications will modulate otherproperties of the iRNA agent as well, e.g., the ability to interact witha protein, e.g., a transport protein, e.g., serum albumin, or a memberof the RISC (RNA-induced Silencing Complex), or the ability of the firstand second sequences to form a duplex with one another or to form aduplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed thatmodifications of the sugar, base, and/or phosphate backbone in an iRNAagent can enhance endonuclease and exonuclease resistance, and canenhance interactions with transporter proteins and one or more of thefunctional components of the RISC complex. Preferred modifications arethose that increase exonuclease and endonuclease resistance and thusprolong the half-life of the iRNA agent prior to interaction with theRISC complex, but at the same time do not render the iRNA agentresistant to endonuclease activity in the RISC complex. Again, while notwishing to be bound by any theory, it is believed that placement of themodifications at or near the 3′ and/or 5′ end of antisense strands canresult in iRNA agents that meet the preferred nuclease resistancecriteria delineated above. Again, still while not wishing to be bound byany theory, it is believed that placement of the modifications at e.g.,the middle of a sense strand can result in iRNA agents that arerelatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into any RNA andRNA-like molecule described herein, e.g., an iRNA agent, a carrieroligonucleotide. An iRNA agent may include a duplex comprising ahybridized sense and antisense strand, in which the antisense strandand/or the sense strand may include one or more of the modificationsdescribed herein. The anti sense strand may include modifications at the3′ end and/or the 5′ end and/or at one or more positions that occur 1-6(e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand.The sense strand may include modifications at the 3′ end and/or the 5′end and/or at any one of the intervening positions between the two endsof the strand. The iRNA agent may also include a duplex comprising twohybridized antisense strands. The first and/or the second antisensestrand may include one or more of the modifications described herein.Thus, one and/or both antisense strands may include modifications at the3′ end and/or the 5′ end and/or at one or more positions that occur 1-6(e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand.Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet thepreferred nuclease resistance criteria delineated above can include oneor more of the following chemical and/or stereochemical modifications ofthe sugar, base, and/or phosphate backbone:

-   -   (i) chiral (S_(P)) thioates. Thus, preferred NRMs include        nucleotide dimers with an enriched or pure for a particular        chiral form of a modified phosphate group containing a        heteroatom at the nonbridging position, e.g., Sp or Rp, where        this is the position normally occupied by the oxygen. The        heteroatom can be S, Se, NR₂, or BR₃. When the heteroatom is S,        enriched or chirally pure Sp linkage is preferred. Enriched        means at least 70, 80, 90, 95, or 99% of the preferred form.        Such NRMs are discussed in more detail below;    -   (ii) attachment of one or more cationic groups to the sugar,        base, and/or the phosphorus atom of a phosphate or modified        phosphate backbone moiety. Thus, preferred NRMs include        compounds at the terminal position derivatized at a cationic        group. As the 5′ end of an antisense sequence should have a        terminal —OH or phosphate group this NRM is preferably not used        at the 5′ end of an anti-sense sequence. The group should be        attached at a position on the base which minimizes interference        with H bond formation and hybridization, e.g., away form the        face which interacts with the complementary base on the other        strand, e.g, at the 5′ position of a pyrimidine or a 7-position        of a purine. These are discussed in more detail below;    -   (iii) nonphosphate linkages at the termini. Thus, preferred NRMs        include Non-phosphate linkages, e.g., a linkage of 4 atoms which        confers greater resistance to cleavage than does a phosphate        bond. Examples include 3′ CH₂—NCH₃—O—CH₂-5′ and 3′        CH₂—NH—(O═C)—CH₂-5′;    -   (iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates.        Thus, preferred NRM's can included these structures;    -   (v) L-RNA, 2′-5′ linkages, inverted linkages, a-nucleosides.        Thus, other preferred NRM's include: L nucleosides and dimeric        nucleotides derived from L-nucleosides; 2′-5′ phosphate,        non-phosphate and modified phosphate linkages (e.g.,        thiophosphates, phosphoramidates and boronophosphates); dimers        having inverted linkages, e.g., 3′-3′ or 5′-5′ linkages;        compounds having an alpha linkage at the 1′ site on the sugar,        e.g., the structures described herein having an alpha linkage;    -   (vi) conjugate groups. Thus, preferred NRM's can include e.g., a        targeting moiety or a conjugated ligand described herein        conjugated with the compound, e.g., through the sugar, base, or        backbone;    -   (vii) abasic linkages. Thus, preferred NRM's can include an        abasic compound, e.g., an abasic compound as described herein        (e.g., a nucleobaseless compound); an aromatic or heterocyclic        or polyheterocyclic aromatic compound as described herein; and    -   (viii) 5′-phosphonates and 5′-phosphate prodrugs. Thus,        preferred NRM's include compounds, preferably at the terminal        position, e.g., the 5′ position, in which one or more atoms of        the phosphate group is derivatized with a protecting group,        which protecting group or groups, are removed as a result of the        action of a component in the subject's body, e.g, a        carboxyesterase or an enzyme present in the subject's body.        E.g., a phosphate prodrug in which a carboxy esterase cleaves        the protected molecule resulting in the production of a thioate        anion which attacks a carbon adjacent to the O of a phosphate        and resulting in the production of an unprotected phosphate.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent. As some NRM'sinterfere with hybridization the total number incorporated, should besuch that acceptable levels of iRNA agent duplex formation aremaintained.

In some embodiments NRM modifications are introduced into the terminalthe cleavage site or in the cleavage region of a sequence (a sensestrand or sequence) which does not target a desired sequence or gene inthe subject. This can reduce off-target silencing.

References General References

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be synthesized with solid phase synthesis, seefor example “Oligonucleotide synthesis, a practical approach”, Ed. M. J.Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A PracticalApproach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1,Modern machine-aided methods of oligodeoxyribonucleotide synthesis,Chapter 2, Oligoribonucleotide synthesis, Chapter 3,2′-O-Methyloligoribonucleotides: synthesis and applications, Chapter 4,Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WO02/44321 can beused herein.

The disclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001,66, 2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 1 1972, 1991. Carbamate replacements are described in Stirchak,E. P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Bases References

N-2 substituted purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.Additional references are disclosed in the above section on basemodifications.

Oligonucleotide Production

The oligonucleotide compounds of the invention can be prepared usingsolution-phase or solid-phase organic synthesis. Organic synthesisoffers the advantage that the oligonucleotide strands comprisingnon-natural or modified nucleotides can be easily prepared. Any othermeans for such synthesis known in the art may additionally oralternatively be employed. It is also known to use similar techniques toprepare other oligonucleotides, such as the phosphorothioates,phosphorodithioates and alkylated derivatives. The double-strandedoligonucleotide compounds of the invention may be prepared using atwo-step procedure. First, the individual strands of the double-strandedmolecule are prepared separately. Then, the component strands areannealed.

Regardless of the method of synthesis, the oligonucleotide can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the iRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried iRNA can then be resuspended in a solutionappropriate for the intended formulation process.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. patents or pendingpatent applications: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn topolyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn tocompounds for the preparation of oligonucleotides having chiralphosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn tooligonucleotides having modified backbones; U.S. Pat. No. 5,386,023,drawn to backbone-modified oligonucleotides and the preparation thereofthrough reductive coupling; U.S. Pat. No. 5,457,191, drawn to modifiednucleobases based on the 3-deazapurine ring system and methods ofsynthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having.beta.-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and5,610,289, drawn to backbone-modified oligonucleotide analogs; and U.S.Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods ofsynthesizing 2′-fluoro-oligonucleotides.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one type of modification maybe incorporated in a single oligonucleotide compound or even in a singlenucleotide thereof.

Routes of Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotide ofthe invention, e.g., modified iRNA agents, antisense, antagomirs,apatamers, ribozymes, and such practice is within the invention. Acomposition that includes an iRNA can be delivered to a subject by avariety of routes. Exemplary routes include: intravenous, topical,rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically include one or more species of iRNA and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the iRNA in aerosol form. The vascularendothelial cells could be targeted by coating a balloon catheter withthe iRNA and mechanically introducing the DNA.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water, syrups, elixirs or non-aqueous media,tablets, capsules, lozenges, or troches. In the case of tablets,carriers that can be used include lactose, sodium citrate and salts ofphosphoric acid. Various disintegrants such as starch, and lubricatingagents such as magnesium stearate, sodium lauryl sulfate and talc, arecommonly used in tablets. For oral administration in capsule form,useful diluents are lactose and high molecular weight polyethyleneglycols. When aqueous suspensions are required for oral use, the nucleicacid compositions can be combined with emulsifying and suspendingagents. If desired, certain sweetening and/or flavoring agents can beadded.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

For ocular administration, ointments or droppable liquids may bedelivered by ocular delivery systems known to the art such asapplicators or eye droppers. Such compositions can include mucomimeticssuch as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbicacid, EDTA or benzylchronium chloride, and the usual quantities ofdiluents and/or carriers.

Topical Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, apatamer,antagomir and ribozyme, and such practice is within the invention. In apreferred embodiment, an iRNA agent is delivered to a subject viatopical administration. “Topical administration” refers to the deliveryto a subject by contacting the formulation directly to a surface of thesubject. The most common form of topical delivery is to the skin, but acomposition disclosed herein can also be directly applied to othersurfaces of the body, e.g., to the eye, a mucous membrane, to surfacesof a body cavity or to an internal surface. As mentioned above, the mostcommon topical delivery is to the skin. The term encompasses severalroutes of administration including, but not limited to, topical andtransdermal. These modes of administration typically include penetrationof the skin's permeability barrier and efficient delivery to the targettissue or stratum. Topical administration can be used as a means topenetrate the epidermis and dermis and ultimately achieve systemicdelivery of the composition. Topical administration can also be used asa means to selectively deliver oligonucleotides to the epidermis ordermis of a subject, or to specific strata thereof, or to an underlyingtissue.

The term “skin,” as used herein, refers to the epidermis and/or dermisof an animal. Mammalian skin consists of two major, distinct layers. Theouter layer of the skin is called the epidermis. The epidermis iscomprised of the stratum corneum, the stratum granulosum, the stratumspinosum, and the stratum basale, with the stratum corneum being at thesurface of the skin and the stratum basale being the deepest portion ofthe epidermis. The epidermis is between 50 μm and 0.2 mm thick,depending on its location on the body.

Beneath the epidermis is the dermis, which is significantly thicker thanthe epidermis. The dermis is primarily composed of collagen in the formof fibrous bundles. The collagenous bundles provide support for, interalia, blood vessels, lymph capillaries, glands, nerve endings andimmunologically active cells.

One of the major functions of the skin as an organ is to regulate theentry of substances into the body. The principal permeability barrier ofthe skin is provided by the stratum corneum, which is formed from manylayers of cells in various states of differentiation. The spaces betweencells in the stratum corneum is filled with different lipids arranged inlattice-like formations that provide seals to further enhance the skinspermeability barrier.

The permeability barrier provided by the skin is such that it is largelyimpermeable to molecules having molecular weight greater than about 750Da. For larger molecules to cross the skin's permeability barrier,mechanisms other than normal osmosis must be used.

Several factors determine the permeability of the skin to administeredagents. These factors include the characteristics of the treated skin,the characteristics of the delivery agent, interactions between both thedrug and delivery agent and the drug and skin, the dosage of the drugapplied, the form of treatment, and the post treatment regimen. Toselectively target the epidermis and dermis, it is sometimes possible toformulate a composition that comprises one or more penetration enhancersthat will enable penetration of the drug to a preselected stratum.

Transdermal delivery is a valuable route for the administration of lipidsoluble therapeutics. The dermis is more permeable than the epidermisand therefore absorption is much more rapid through abraded, burned ordenuded skin. Inflammation and other physiologic conditions thatincrease blood flow to the skin also enhance transdermal adsorption.Absorption via this route may be enhanced by the use of an oily vehicle(inunction) or through the use of one or more penetration enhancers.Other effective ways to deliver a composition disclosed herein via thetransdermal route include hydration of the skin and the use ofcontrolled release topical patches. The transdermal route provides apotentially effective means to deliver a composition disclosed hereinfor systemic and/or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biologicalmembranes under the influence of an electric field) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163),phonophoresis or sonophoresis (use of ultrasound to enhance theabsorption of various therapeutic agents across biological membranes,notably the skin and the cornea) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 166), and optimization ofvehicle characteristics relative to dose position and retention at thesite of administration (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 168) may be useful methods for enhancing thetransport of topically applied compositions across skin and mucosalsites.

The compositions and methods provided may also be used to examine thefunction of various proteins and genes in vitro in cultured or preserveddermal tissues and in animals. The invention can be thus applied toexamine the function of any gene. The methods of the invention can alsobe used therapeutically or prophylactically. For example, for thetreatment of animals that are known or suspected to suffer from diseasessuch as psoriasis, lichen planus, toxic epidermal necrolysis, ertythemamultiforme, basal cell carcinoma, squamous cell carcinoma, malignantmelanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lymedisease and viral, fungal and bacterial infections of the skin.

Pulmonary Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, apatamer,antagomir and ribozyme, and such practice is within the invention. Acomposition that includes an iRNA agent, e.g., a double-stranded iRNAagent, can be administered to a subject by pulmonary delivery. Pulmonarydelivery compositions can be delivered by inhalation by the patient of adispersion so that the composition, preferably iRNA, within thedispersion can reach the lung where it can be readily absorbed throughthe alveolar region directly into blood circulation. Pulmonary deliverycan be effective both for systemic delivery and for localized deliveryto treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An iRNAcomposition may be stably stored as lyophilized or spray-dried powdersby itself or in combination with suitable powder carriers. The deliveryof a composition for inhalation can be mediated by a dosing timingelement which can include a timer, a dose counter, time measuringdevice, or a time indicator which when incorporated into the deviceenables dose tracking, compliance monitoring, and/or dose triggering toa patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersedsolid particles that are free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a subjectso that the particles reach the lungs to permit penetration into thealveoli. Thus, the powder is said to be “respirable.” Preferably theaverage particle size is less than about 10 μm in diameter preferablywith a relatively uniform spheroidal shape distribution. More preferablythe diameter is less than about 7.5 μm and most preferably less thanabout 5.0 μm. Usually the particle size distribution is between about0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5μm.

The term “dry” means that the composition has a moisture content belowabout 10% by weight (% w) water, usually below about 5% w and preferablyless it than about 3% w. A dry composition can be such that theparticles are readily dispersible in an inhalation device to form anaerosol.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Additives, which are minor components of the composition of thisinvention, may be included for conformational stability during spraydrying and for improving dispersibility of the powder. These additivesinclude hydrophobic amino acids such as tryptophan, tyrosine, leucine,phenylalanine, and the like.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

Pulmonary administration of a micellar iRNA formulation may be achievedthrough metered dose spray devices with propellants such astetrafluoroethane, heptafluoroethane, dimethylfluoropropane,tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFCand CFC propellants.

Oral or Nasal Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, apatamer,antagomir and ribozyme, and such practice is within the invention. Boththe oral and nasal membranes offer advantages over other routes ofadministration. For example, drugs administered through these membraneshave a rapid onset of action, provide therapeutic plasma levels, avoidfirst pass effect of hepatic metabolism, and avoid exposure of the drugto the hostile gastrointestinal (GI) environment. Additional advantagesinclude easy access to the membrane sites so that the drug can beapplied, localized and removed easily.

In oral delivery, compositions can be targeted to a surface of the oralcavity, e.g., to sublingual mucosa which includes the membrane ofventral surface of the tongue and the floor of the mouth or the buccalmucosa which constitutes the lining of the cheek. The sublingual mucosais relatively permeable thus giving rapid absorption and acceptablebioavailability of many drugs. Further, the sublingual mucosa isconvenient, acceptable and easily accessible.

The ability of molecules to permeate through the oral mucosa appears tobe related to molecular size, lipid solubility and peptide proteinionization. Small molecules, less than 1000 daltons appear to crossmucosa rapidly. As molecular size increases, the permeability decreasesrapidly. Lipid soluble compounds are more permeable than non-lipidsoluble molecules. Maximum absorption occurs when molecules areun-ionized or neutral in electrical charges. Therefore charged moleculespresent the biggest challenges to absorption through the oral mucosae.

A pharmaceutical composition of iRNA may also be administered to thebuccal cavity of a human being by spraying into the cavity, withoutinhalation, from a metered dose spray dispenser, a mixed micellarpharmaceutical formulation as described above and a propellant. In oneembodiment, the dispenser is first shaken prior to spraying thepharmaceutical formulation and propellant into the buccal cavity.

Devices

For ease of exposition the devices, formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, apatamer,antagomir and ribozyme, and such practice is within the invention. AniRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof) can be disposed on orin a device, e.g., a device which implanted or otherwise placed in asubject. Exemplary devices include devices which are introduced into thevasculature, e.g., devices inserted into the lumen of a vascular tissue,or which devices themselves form a part of the vasculature, includingstents, catheters, heart valves, and other vascular devices. Thesedevices, e.g., catheters or stents, can be placed in the vasculature ofthe lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted inthe peritoneum, or in organ or glandular tissue, e.g., artificialorgans. The device can release a therapeutic substance in addition to aiRNA, e.g., a device can release insulin.

Other devices include artificial joints, e.g., hip joints, and otherorthopedic implants.

In one embodiment, unit doses or measured doses of a composition thatincludes iRNA are dispensed by an implanted device. The device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include pump, e.g., and, optionally, associatedelectronics.

Tissue, e.g., cells or organs, such as the kidney, can be treated withan iRNA agent ex vivo and then administered or implanted in a subject.

The tissue can be autologous, allogeneic, or xenogeneic tissue. Forexample, tissue (e.g., kidney) can be treated to reduce graft v. hostdisease. In other embodiments, the tissue is allogeneic and the tissueis treated to treat a disorder characterized by unwanted gene expressionin that tissue, such as in the kidney. In another example, tissuecontaining hematopoietic cells, e.g., bone marrow hematopoietic cells,can be treated to inhibit unwanted cell proliferation.

Introduction of treated tissue, whether autologous or transplant, can becombined with other therapies.

In some implementations, the iRNA treated cells are insulated from othercells, e.g., by a semi-permeable porous barrier that prevents the cellsfrom leaving the implant, but enables molecules from the body to reachthe cells and molecules produced by the cells to enter the body. In oneembodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains aniRNA agent. Exemplary devices include condoms, diaphragms, IUD(implantable uterine devices, sponges, vaginal sheaths, and birthcontrol devices. In one embodiment, the iRNA is chosen to inactive spermor egg. In another embodiment, the iRNA is chosen to be complementary toa viral or pathogen RNA, e.g., an RNA of an STD. In some instances, theiRNA composition can include a spermicidal agent.

Formulations

The iRNA agents described herein can be formulated for administration toa subject. For ease of exposition the formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It should be understood, however, that these formulations,compositions and methods can be practiced with other oligonucleotides ofthe invention, e.g., modified iRNA agents, antisense, apatamer,antagomir and ribozyme, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration.

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

An iRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent,e.g., a second iRNA that can mediated RNAi with respect to a secondgene, or with respect to the same gene. Still other preparation caninclude at least 3, 5, ten, twenty, fifty, or a hundred or moredifferent iRNA species. Such iRNAs can mediated RNAi with respect to asimilar number of different genes.

In one embodiment, the iRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than a RNA or a DNA). Forexample, an iRNA composition for the treatment of a viral disease, e.g.HIV, might include a known antiviral agent (e.g., a protease inhibitoror reverse transcriptase inhibitor). In another example, an iRNAcomposition for the treatment of a cancer might further comprise achemotherapeutic agent.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Pharmaceutical Compositions

In one embodiment, the invention relates to a pharmaceutical compositioncontaining an oligonucleotide of the invention e.g. an iRNA agent, asdescribed in the preceding sections, and a pharmaceutically acceptablecarrier, as described below. A pharmaceutical composition including themodified iRNA agent is useful for treating a disease caused byexpression of a target gene. In this aspect of the invention, the iRNAagent of the invention is formulated as described below. Thepharmaceutical composition is administered in a dosage sufficient toinhibit expression of the target gene.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit the expression or activityof the target gene. Compositions containing the iRNA agent of theinvention can be administered at surprisingly low dosages. A maximumdosage of 5 mg iRNA agent per kilogram body weight per day may besufficient to inhibit or completely suppress the expression or activityof the target gene.

In general, a suitable dose of modified iRNA agent will be in the rangeof 0.001 to 500 milligrams per kilogram body weight of the recipient perday (e.g., about 1 microgram per kilogram to about 500 milligrams perkilogram, about 100 micrograms per kilogram to about 100 milligrams perkilogram, about 1 milligrams per kilogram to about 75 milligrams perkilogram, about 10 micrograms per kilogram to about 50 milligrams perkilogram, or about 1 microgram per kilogram to about 50 micrograms perkilogram). The pharmaceutical composition may be administered once perday, or the iRNA agent may be administered as two, three, four, five,six or more sub-doses at appropriate intervals throughout the day. Inthat case, the iRNA agent contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage. Thedosage unit can also be compounded for delivery over several days, e.g.,using a conventional sustained release formulation which providessustained release of the iRNA agent over a several day period. Sustainedrelease formulations are well known in the art. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the infection or disease, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNA agent encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse repositories canbe found at The Jackson Laboratory, Charles River Laboratories, Taconic,Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Networkand at the European Mouse Mutant Archive. Such models may be used for invivo testing of iRNA agent, as well as for determining a therapeuticallyeffective dose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), ocular,rectal, vaginal and topical (including buccal and sublingual)administration. In preferred embodiments, the pharmaceuticalcompositions are administered by intravenous or intraparenteral infusionor injection. The pharmaceutical compositions can also be administeredintraparenchymally, intrathecally, and/or by stereotactic injection.

For oral administration, the iRNA agent useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredient is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the pharmaceutical compositions of the invention will generally beprovided in sterile aqueous solutions or suspensions, buffered to anappropriate pH and isotonicity. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of iRNAagent in the cells that harbor the target gene or virus. Such substancesinclude, for example, micellar structures, such as liposomes or capsids,as described below. Although microinjection, lipofection, viruses,viroids, capsids, capsoids, or other auxiliary agents are required tointroduce iRNA agent into cell cultures, surprisingly these methods andagents are not necessary for uptake of iRNA agent in vivo. The iRNAagent of the present invention are particularly advantageous in thatthey do not require the use of an auxiliary agent to mediate uptake ofthe iRNA agent into the cell, many of which agents are toxic orassociated with deleterious side effects. Aqueous suspensions accordingto the invention may include suspending agents such as cellulosederivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth,and a wetting agent such as lecithin. Suitable preservatives for aqueoussuspensions include ethyl and n-propyl p-hydroxybenzoate.

The pharmaceutical compositions can also include encapsulatedformulations to protect the iRNA agent against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811; PCT publication WO 91/06309; and European patent publicationEP-A-43075, which are incorporated by reference herein.

Toxicity and therapeutic efficacy of iRNA agent can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.iRNA agents that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosages ofcompositions of the invention are preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyiRNA agent used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the iRNA agent or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test iRNA agent which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, iRNA agents relating to the invention can beadministered in combination with other known agents effective intreating viral infections and diseases. In any event, the administeringphysician can adjust the amount and timing of iRNA agent administrationon the basis of results observed using standard measures of efficacyknown in the art or described herein.

Combination Therapy

In one aspect, composition of the invention can be used in combinationtherapy. The term “combination therapy” includes the administration ofthe subject compounds in further combination with other biologicallyactive ingredients (such as, but not limited to, a second and differentantineoplastic agent) and non-drug therapies (such as, but not limitedto, surgery or radiation treatment). For instance, the compounds of theinvention can be used in combination with other pharmaceutically activecompounds, preferably compounds that are able to enhance the effect ofthe compounds of the invention. The compounds of the invention can beadministered simultaneously (as a single preparation or separatepreparation) or sequentially to the other drug therapy. In general, acombination therapy envisions administration of two or more drugs duringa single cycle or course of therapy.

In one aspect of the invention, the subject compounds may beadministered in combination with one or more separate agents thatmodulate protein kinases involved in various disease states. Examples ofsuch kinases may include, but are not limited to: serine/threoninespecific kinases, receptor tyrosine specific kinases and non-receptortyrosine specific kinases. Serine/threonine kinases include mitogenactivated protein kinases (MAPK), meiosis specific kinase (MEK), RAF andaurora kinase. Examples of receptor kinase families include epidermalgrowth factor receptor (EGFR) (e.g. HER2/neu, HER3, HER4, ErbB, ErbB2,ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor(e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R);hepatocyte growth/scatter factor receptor (HGFR) (e.g, MET, RON, SEA,SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK,EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g.Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR)(e.g. PDGFα-R, PDGβ-R, CSFl-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1,FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but arenot limited to, BCR-ABL (e.g. p43^(abl), ARG); BTK (e.g. ITK/EMT, TEC);CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may beadministered in combination with one or more agents that modulatenon-kinase biological targets or processes. Such targets include histonedeacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins(e.g. HSP90), and proteosomes.

In one embodiment, subject compounds may be combined with antineoplasticagents (e.g. small molecules, monoclonal antibodies, antisense RNA, andfusion proteins) that inhibit one or more biological targets such asZolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar,Sorafinib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin,Erbitux, AG24322, PD325901, ZD6474, PD 184322,

Obatodax, ABT737 and AEE788. Such combinations may enhance therapeuticefficacy over efficacy achieved by any of the agents alone and mayprevent or delay the appearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents encompass a wide range of therapeutic treatmentsin the field of oncology. These agents are administered at variousstages of the disease for the purposes of shrinking tumors, destroyingremaining cancer cells left over after surgery, inducing remission,maintaining remission and/or alleviating symptoms relating to the canceror its treatment. Examples of such agents include, but are not limitedto, alkylating agents such as mustard gas derivatives

(Mechlorethamine, cylophosphamide, chlorambucil, melphalan, ifosfamide),ethylenimines (thiotepa, hexamethylmelanine), Alkylsulfonates(Busulfan), Hydrazines and Triazines (Altretamine, Procarbazine,Dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine andStreptozocin), Ifosfamide and metal salts (Carboplatin, Cisplatin, andOxaliplatin); plant alkaloids such as Podophyllotoxins (Etoposide andTenisopide), Taxanes (Paclitaxel and Docetaxel), Vinca alkaloids(Vincristine, Vinblastine, Vindesine and Vinorelbine), and Camptothecananalogs (Irinotecan and Topotecan); anti-tumor antibiotics such asChromomycins (Dactinomycin and Plicamycin), Anthracyclines (Doxorubicin,Daunorubicin, Epirubicin, Mitoxantrone, Valrubicin and Idarubicin), andmiscellaneous antibiotics such as Mitomycin, Actinomycin and Bleomycin;anti-metabolites such as folic acid antagonists (Methotrexate,Pemetrexed, Raltitrexed, Aminopterin), pyrimidine antagonists(5-Fluorouracil, Floxuridine, Cytarabine, Capecitabine, andGemcitabine), purine antagonists (6-Mercaptopurine and 6-Thioguanine)and adenosine deaminase inhibitors (Cladribine, Fludarabine,Mercaptopurine, Clofarabine, Thioguanine, Nelarabine and Pentostatin);topoisomerase inhibitors such as topoisomerase I inhibitors (Ironotecan,topotecan) and topoisomerase II inhibitors (Amsacrine, etoposide,etoposide phosphate, teniposide); monoclonal antibodies (Alemtuzumab,Gemtuzumab ozogamicin, Rituximab, Trastuzumab, Ibritumomab Tioxetan,Cetuximab, Panitumumab, Tositumomab,

Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotidereductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor(Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubuleagents (Estramustine); and retinoids (Bexarotene, Isotretinoin,Tretinoin (ATRA). In certain preferred embodiments, the compounds of theinvention are administered in combination with a chemoprotective agent.Chemoprotective agents act to protect the body or minimize the sideeffects of chemotherapy. Examples of such agents include, but are notlimited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administeredin combination with radiation therapy. Radiation is commonly deliveredinternally (implantation of radioactive material near cancer site) orexternally from a machine that employs photon (x-ray or gamma-ray) orparticle radiation. Where the combination therapy further comprisesradiation treatment, the radiation treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and radiation treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the radiation treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

It will be appreciated that compounds of the invention can be used incombination with an immunotherapeutic agent. One form of immunotherapyis the generation of an active systemic tumor-specific immune responseof host origin by administering a vaccine composition at a site distantfrom the tumor. Various types of vaccines have been proposed, includingisolated tumor-antigen vaccines and anti-idiotype vaccines. Anotherapproach is to use tumor cells from the subject to be treated, or aderivative of such cells (reviewed by Schirrmacher et al. (1995) J.Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr.et al. claim a method for treating a respectable carcinoma to preventrecurrence or metastases, comprising surgically removing the tumor,dispersing the cells with collagenase, irradiating the cells, andvaccinating the patient with at least three consecutive doses of about10⁷ cells.

It will be appreciated that the compounds of the invention mayadvantageously be used in conjunction with one or more adjunctivetherapeutic agents. Examples of suitable agents for adjunctive therapyinclude steroids, such as corticosteroids (amcinonide, betamethasone,betamethasone dipropionate, betamethasone valerate, budesonide,clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol17-propionate, cortisone, deflazacort, desoximetasone, diflucortolonevalerate, dexamethasone, dexamethasone sodium phosphate, desonide,furoate, fluocinonide, fluocinolone acetonide, halcinonide,hydrocortisone, hydrocortisone butyrate, hydrocortisone sodiumsuccinate, hydrocortisone valerate, methyl prednisolone, mometasone,prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, andhalobetasol proprionate); a 5HTi agonist, such as a triptan (e.g.sumatriptan or naratriptan); an adenosine Al agonist; an EP ligand; anNMDA modulator, such as a glycine antagonist; a sodium channel blocker(e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); acannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; aleukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentinand related compounds; a tricyclic antidepressant (e.g. amitryptilline);a neurone stabilising antiepileptic drug; a mono-aminergic uptakeinhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; anitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOSinhibitor; an inhibitor of the release, or action, of tumour necrosisfactor c; an antibody therapy, such as a monoclonal antibody therapy; anantiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or animmune system modulator (e.g. interferon); an opioid analgesic; a localanaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g.ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g.aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); adecongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine,oxymetazoline, epinephrine, naphazoline, xylometazoline,propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine,hydrocodone, carmiphen, carbetapentane, or dextramethorphan); adiuretic; or a sedating or non-sedating antihistamine.

Methods for Inhibiting Expression of a Target Gene

In yet another aspect, the invention relates to a method for inhibitingthe expression of a target gene in a cell or organism. In oneembodiment, the method includes administering the inventiveoligonucleotide, e.g. antisense, aptamer, antagomir, or an iRNA agent;or a pharmaceutical composition containing the said oligonucleotide to acell or an organism, such as a mammal, such that expression of thetarget gene is silenced. Compositions and methods for inhibiting theexpression of a target gene using the inventive oligonucleotide, e.g. aniRNA agent, can be performed as described in the preceding sections.

In this embodiment, a pharmaceutical composition containing theinventive oligonucleotide may be administered by any means known in theart including, but not limited to oral or parenteral routes, includingintravenous, intramuscular, intraperitoneal, subcutaneous, transdermal,airway (aerosol), ocular, rectal, vaginal, and topical (including buccaland sublingual) administration. In preferred embodiments, thepharmaceutical compositions are administered by intravenous orintraparenteral infusion or injection. The pharmaceutical compositionscan also be administered intraparenchymally, intrathecally, and/or bystereotactic injection.

The methods for inhibiting the expression of a target gene can beapplied to any gene one wishes to silence, thereby specificallyinhibiting its expression, provided the cell or organism in which thetarget gene is expressed includes the cellular machinery which effectsRNA interference. Examples of genes which can be targeted for silencinginclude, without limitation, developmental genes including but notlimited to adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, andneurotransmitters and their receptors; (2) oncogenes including but notlimited to ABLI, BCL1, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2,ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2,MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 andYES; (3) tumor suppresser genes including but not limited to APC, BRCA1,BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53 and WT1; and (4) enzymesincluding but not limited to ACP desaturases and hydroxylases,ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, cyclooxygenases,decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases,glucanases, glucose oxidases, GTPases, helicases, hemicellulases,integrases, invertases, isomerases, kinases, lactases, lipases,lipoxygenases, lysozymes, pectinesterases, peroxidases, phosphatases,phospholipases, phosphorylases, polygalacturonases, proteinases andpeptideases, pullanases, recombinases, reverse transcriptases,topoisomerases, and xylanases.

In addition to in vivo gene inhibition, the skilled artisan willappreciate that the inventive oligonucleotides, e.g. iRNA agent, of thepresent invention are useful in a wide variety of in vitro applications.Such in vitro applications, include, for example, scientific andcommercial research (e.g., elucidation of physiological pathways, drugdiscovery and development), and medical and veterinary diagnostics. Ingeneral, the method involves the introduction of the oligonucleotide,e.g. an iRNA agent, into a cell using known techniques (e.g., absorptionthrough cellular processes, or by auxiliary agents or devices, such aselectroporation and lipofection), then maintaining the cell for a timesufficient to obtain degradation of an mRNA transcript of the targetgene.

DEFINITIONS

The term “aliphatic,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms whereinthe saturation between any two carbon atoms is a single, double ortriple bond. An aliphatic group preferably contains from 1 to about 24carbon atoms, more typically from 1 to about 12 carbon atoms. Suitablealiphatic groups include, but are not limited to, linear or branched,substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybridsthereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl. The straight or branched chain of an aliphaticgroup may be interrupted with one or more heteroatoms that includenitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines, for example.Aliphatic groups as used herein may optionally include furthersubstitutent groups.

The term “alkyl” refers to saturated and unsaturated non-aromatichydrocarbon chains that may be a straight chain or branched chain,containing the indicated number of carbon atoms (these include withoutlimitation propyl, allyl, or propargyl), which may be optionallyinserted with N, O, or S. For example, C₁-C₂₀ indicates that the groupmay have from 1 to 20 (inclusive) carbon atoms in it. The term “alkoxy”refers to an —O-alkyl radical. The term “alkylene” refers to a divalentalkyl (i.e., —R—). The term “alkylenedioxo” refers to a divalent speciesof the structure —O—R—O—, in which R represents an alkylene. The term“aminoalkyl” refers to an alkyl substituted with an amino. The term“mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an—S-alkyl radical.

The term “cyclic” as used herein includes a cycloalkyl group and aheterocyclic group. Any suitable ring position of the cyclic group maybe covalently linked to the defined chemical structure.

The term “acyclic” may describe any carrier that is branched orunbranched, and does not form a closed ring.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Cycloalkyl groups include, without limitation, decalin, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocycloalkyl” and “heterocyclic” can be usedinterchangeably and refer to a non-aromatic 3-, 4-, 5-, 6- or 7-memberedring or a bi- or tri-cyclic group fused system, where (i) each ringcontains between one and three heteroatoms independently selected fromoxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 doublebonds and each 6-membered ring has 0 to 2 double bonds, (iii) thenitrogen and sulfur heteroatoms may optionally be oxidized, (iv) thenitrogen heteroatom may optionally be quaternized, (iv) any of the aboverings may be fused to a benzene ring, and (v) the remaining ring atomsare carbon atoms which may be optionally oxo-substituted. Representativeheterocycloalkyl groups include, but are not limited to, [1,3]dioxolane,pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, andtetrahydrofuryl. Such heterocyclic groups may be further substituted togive substituted heterocyclic.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen or a substituted orunsubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl,arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl,alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl,arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino,trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl,arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl,carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl,heteroaryl, heterocyclic, and aliphatic. It is understood that thesubstituent may be further substituted.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the above basesand “universal bases” can be employed. Examples include 2-aminoadenine,2-fluoroadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

The term “non-natural” nucleobase refers any one of the following:2-methyladeninyl, N6-methyladeninyl, 2-methylthio-N6-methyladeninyl,N6-isopentenyladeninyl, 2-methylthio-N6-isopentenyladeninyl,N6-(cis-hydroxyisopentenyl)adeninyl,2-methylthio-N6-(cis-hydroxyisopentenyl)adeninyl,N6-glycinylcarbamoyladeninyl, N6-threonylcarbamoyladeninyl,2-methylthio-N6-threonyl carbamoyladeninyl,N6-methyl-N6-threonylcarbamoyladeninyl,N6-hydroxynorvalylcarbamoyladeninyl, 2-methylthio-N6-hydroxynorvalylcarbamoyladeninyl, N6,N6-dimethyladeninyl, 3-methylcytosinyl,5-methylcytosinyl, 2-thiocytosinyl, 5-formylcytosinyl,N4-methylcytosinyl, 5-hydroxymethylcytosinyl, 1-methylguaninyl,N2-methylguaninyl, 7-methylguaninyl, N2,N2-dimethylguaninyl,N2,7-dimethylguaninyl, N2,N2,7-trimethylguaninyl, 1-methylguaninyl,7-cyano-7-deazaguaninyl, 7-aminomethyl-7-deazaguaninyl, pseudouracilyl,dihydrouracilyl, 5-methyluracilyl, 1-methylpseudouracilyl,2-thiouracilyl, 4-thiouracilyl, 2-thiothyminyl 5-methyl-2-thiouracilyl,3-(3-amino-3-carboxypropyl)uracilyl, 5-hydroxyuracilyl,5-methoxyuracilyl, uracilyl 5-oxyacetic acid, uracilyl 5-oxyacetic acidmethyl ester, 5-(carboxyhydroxymethyl)uracilyl,5-(carboxyhydroxymethyl)uracilyl methyl ester,5-methoxycarbonylmethyluracilyl, 5-methoxycarbonylmethyl-2-thiouracilyl,5-aminomethyl-2-thiouracilyl, 5-methylaminomethyluracilyl,5-methylaminomethyl-2-thiouracilyl,5-methylaminomethyl-2-selenouracilyl, 5-carbamoylmethyluracilyl,5-carboxymethylaminomethyluracilyl,5-carboxymethylaminomethyl-2-thiouracilyl, 3-methyluracilyl,1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,5-carboxymethyluracilyl, 5-methyldihydrouracilyl,3-methylpseudouracilyl,

A universal base can form base pairs with each of the natural DNA/RNAbases, exhibiting relatively little discrimination between them. Ingeneral, the universal bases are non-hydrogen bonding, hydrophobic,aromatic moieties which can stabilize e.g., duplex RNA or RNA-likemolecules, via stacking interactions. A universal base can also includehydrogen bonding substituents. As used herein, a “universal base” caninclude anthracenes, pyrenes or any one of the following:

Antagomirs

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-O-methylation of sugar, phosphorothioate backboneand, for example, a cholesterol-moiety at 3′-end. Antagomirs may be usedto efficiently silence endogenous miRNAs thereby preventingmiRNA-induced gene silencing. An example of antagomir-mediated miRNAsilencing is the silencing of miR-122, described in Krutzfeldt et al,Nature, 2005, 438: 685-689, which is expressly incorporated by referenceherein, in its entirety.

Decoy Oligonucleotides

Because transcription factors can recognize their relatively shortbinding sequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to upregulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides may be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety.

Antisense Oligonucleotides

Antisense oligonucleotides are single strands of DNA or RNA that are atleast partially complementary to a chosen sequence. In the case ofantisense RNA, they prevent translation of complementary RNA strands bybinding to it. Antisense DNA can also be used to target a specific,complementary (coding or non-coding) RNA. If binding takes place, theDNA/RNA hybrid can be degraded by the enzyme RNase H. Examples of theutilization of antisense oligonucleotides may be found in Dias et al.,Mol. Cancer Ther., 2002, 1: 347-355, which is expressly incorporated byreference herein, in its entirety.

Aptamers

Aptamers are nucleic acid molecules that bind a specific target moleculeor molecules. Aptamers may be RNA or DNA based, and may include ariboswitch. A riboswitch is a part of an mRNA molecule that can directlybind a small target molecule, and whose binding of the target affectsthe gene's activity. Thus, an mRNA that contains a riboswitch isdirectly involved in regulating its own activity, depending on thepresence or absence of its target molecule.

REFERENCES

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. It is, therefore, to beunderstood that the foregoing embodiments are presented by way ofexample only and that, within the scope of the appended claims andequivalents thereto, the invention may be practiced otherwise than asspecifically described and claimed.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the invention, and are not intended to limit the invention. Thus, theinvention should in way be construed as being limited to the followingexamples, but rather, should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

SYNTHETIC SCHEMES AND EXAMPLES

Alkyne derivatives for click chemistry (siRNA conjugation)

Example 1 Synthesis of Alkyne Derivative

Synthesis of Compound 91:

To an ice-cooled solution of phenyl acetaldehyde (200 g, 1.662 mol) indichloromethane (1 L) was added iodotrimethylsilane (475 mL, 3.325 mol)drop-wise. The reaction mass was allowed to reach room temperature andstirred for 16 hours. The reaction was quenched with 1M sodiumthiosulfate solution. The layers were separated. The aqueous layer wasextracted with dichloromethane (1×500 ml). Combined organic layer waswashed with saturated sodium bicarbonate (3×1 L), followed by brinewash. The organic layer was dried and evaporated at reduced pressure toobtain crude product, which was purified by silica gel chromatographyusing ether/hexane as eluent to get the product (70.23 g, 38%) as abrown solid. ¹H NMR (400 MHz, CDCl₃): δ 2.75-2.79 (d, 2H, J=16 Hz),3.52-3.58 (dd, 2H, J=6 Hz), 5.30-5.29 (d, 2H, J=6 Hz) and 6.96-7.50 (m,8H). ¹³C NMR (100 MHz, CDCl₃): δ 36.11, 69.55, 76.68, 77.00, 77.32,125.14, 125.95, 126.81, 128.35, 129.07, 131.59 and 137.78. GC-MS: 222

Synthesis of Compound 92:

Compound 91 (66 g, 0.29 mol) was dissolved in THF (600 mL) and cooled to0-4° C. To the cooled solution was added n-butyl lithium (256 mL, 23% inhexane) drop wise maintaining temperature between 4-10° C. The reactionmass was allowed to reach RT and stirred for 2 hours. The reaction wasquenched slowly with 5 ml of water first, later with excess water. Thelayers were separated and the aqueous layer was extracted withdichloromethane. The combined organic layer was washed with water,followed by brine and dried over sodium sulfate. The organic layer wasevaporated at reduced pressure to obtain crude product, which waspurified by silica gel chromatography using ethyl acetate/hexane aseluent to get the product as a white solid (43 g, 65%). ¹H NMR (400 MHz,CDCl₃): δ 3.29-3.35 (m, 1H), 3.42-3.47 (m, 1H), 5.26-5.30 (m, 1H)6.80-6.88 (m, 2H), 7.09-7.25 (m, 7H) and 7.36-7.46 (m, 1H). ¹³C NMR (100MHz, CDCl₃): δ 42.52, 69.55, 74.27, 125.78, 126.82, 127.04, 127.28,129.22, 129.81, 130.47, 131.40, 134.35, 136.12, 136.66 and 140.69.GC-MS: 222.

Synthesis of Compound 93:

To an ice-cooled solution of product 92 (43 g, 0.194 mol) in DMF (430mL) under argon atmosphere was added imidazole (19.8 g, 0.29 mol)followed by ter-butyldimethylchlorosilane (43.7 g, 0.29 mol). Thereaction mass was allowed to reach room temperature and stirred for onehour. The reaction mass was diluted with water (1 L) and extracted withether. The organic layer was washed with water (700 ml), followed bybrine, dried over sodium sulfate and finally evaporated at reducedpressure to obtain crude product, which was purified by silica gelchromatography using ether/hexane as eluent to get the product as yellowviscous liquid (45 g, 70%) ¹H NMR (400 MHz, CDCl₃): δ 0.063 (s, 3H),0.081 (s, 3H), 0.95 (s, 9H), 3.20-3.24 (m, 1H), 3.50-3.56 (m, 1H),5.48-5.52 (m, 1H) 6.80-6.88 (m, 2H), 7.09-7.25 (m, 7H) and 7.36-7.46 (m,1H).

Synthesis of Compound 94:

To an ice-cooled solution of compound 93 (38 g, 0.113 mol) in chloroform(380 mL) was added bromine (21.70 g, 0.135 mol) in chloroform drop wiseover a period of 40 minutes at 0-4° C. The reaction was quenched, justafter the addition, with saturated sodium thiosulfate and separated thelayers. The aqueous layer was extracted with chloroform (1×500 ml). Thecombined organic layer was washed once with sodium thiosulfate (1×300ml), followed by water (1×300 ml), dried over sodium sulfate and finallyevaporated at reduced pressure to obtain the crude product, which waspurified by silica chromatography using ether/hexane as eluent to getthe product as pale brown liquid (20.7 g, 48%). ¹H NMR (400 MHz, CDCl₃):δ 2.81-3.10 (m, 1H), 3.55-3.75 (m, 1H), 5.27-5.8 (m, 3H) and 6.86-7.58(m, 8H).

Synthesis of Compound 95:

To a solution of compound 94 (110 g, 0.28 mol) in THF (3.3 L), was addedfreshly prepared LDA (92.4 g, 0.86 mol) in THF at room temperature andallowed to stir for half an hour. The reaction was quenched with waterand separated the layers. The aqueous layer was extracted withdichloromethane. The combined organic layer was dried over sodiumsulfate and evaporated at reduced pressure. The crude product obtainedwas purified by silica gel chromatography using ethyl acetate/hexane aseluent to get the product as a white solid (50 g, 79%). ¹H NMR (400 MHz,CDCl₃): δ 2.90-3.12 (m, 2H), 4.6 (s, 1H) and 7.29-7.75 (m, 8H).

Synthesis of 97:

To an ice-cooled solution of compound 95 (50 g, 0.22 mol) indichloromethane (500 mL) was added N,N′-disuccinimidyl carbonate (116 g,0.45 mol), followed by triethyl amine (64.4 mL, 0.46 mol) and allowed toreach room temperature under argon atmosphere. The reaction mass wasallowed to stir for 14 hours at room temperature. Diluted withdichloromethane and washed with water and brine. Dried over sodiumsulfate and removed the solvent. The crude obtained was taken as such tothe next stage without isolating the product (82 g). To an ice-cooledsolution of DSC derivative in dichloromethane (900 mL) was added methyl6-aminocaproate (65 g, 0.45 mol) in dichloromethane, followed bytriethylamine (65 mL, 0.45 mol) and allowed to reach room temperature.The solvents were evaporated to dryness at reduced pressure to get crudeproduct, which was purified by silica gel chromatography usingmethanol/dichloromethane as eluent to get the product as a pale yellowliquid (67.3 g, 60%). ¹H NMR (400 MHz, CDCl₃): δ 1.37-1.41 (m, 2H),1.64-1.67 (m, 2H), 2.30-2.34 (t, 2H, J=18 Hz), 2.87-2.92 (m, 2H),3.13-3.21 (m, 4H), 3.67 (s, 3H), 4.97 (m, 1H), 5.4 (s, 1H) and 7.26-7.49(m, 8H).

Synthesis of 98:

To a solution of compound 97 (45 g, 0.11 mol) in methanol and THF wasadded lithium hydroxide (10.5 g, 0.23 mol) in water at room temperature(480 mL, water:THF:MeOH 2:1:1). The reaction mass was allowed to stirfor half an hour. The solvents were evaporated at reduced pressure. Theresidue obtained was diluted with water and washed with ethyl acetate(3×250 ml) to remove impurities. The aqueous layer then was acidifiedwith 10% hydrochloric acid and extracted with ethyl acetate. The organiclayer was dried over sodium sulfate and evaporated at reduced pressureto afford pure product as pale brown solid (43 g, 99%). ¹H NMR (400 MHz,DMSO-d₆): δ 1.24-1.32 (m, 2H), 1.39-1.54 (m, 4H), 2.18-2.22 (t, 2H, J=16Hz), 2.75-2.79 (dd, 1H, J=9 Hz), 2.96-3.01 (m, 2H), 3.16-3.20 (d, 2H,J=16 Hz), 5.3 (s, 1H) and 7.3-7.6 (m, 8H). ¹³C NMR (100 MHz, DMSO-d₆): δ24.10, 25.70, 28.96, 33.52, 40.07, 45.47, 75.16, 109.84, 112.51, 120.29,122.84, 123.71, 125.68, 126.00, 127.20, 127.25, 128.31, 130.02, 150.81,152.50, 155.14, 171.87, and 174.30. LC-MS: 376 (M−1)⁺.

Example 2 Copper Free Click Chemistry with Azido Derivatives

Synthesis of 100:

Compound 98 (0.500 g, 1.32 mmol) and the GalNAc azide 99 (0.668 g, 1.32mmol) were taken in MeOH (15 mL) and stirred the reaction mixture underargon. Reaction was monitored by TLC, in 2 hrs reaction was complete.Solvent was removed the residue was purified by chromatography (2-5%MeOH/DCM) to get the product as a color less liquid (0.960 g, 85%).1HNMR (DMSO-d6) d=MS calculated for C₄₃H₅₅N₅O₁₅ 881.37. Found 904.36(M+Na).

Synthesis of 102:

Compound 98 (0.500 g, 1.32 mmol) and the mannose azide 101 (0.994 g,1.32 mmol) were taken in MeOH (15 mL) and stirred the reaction mixtureunder argon. Reaction was monitored by TLC, in 3 hrs reaction wascomplete. Solvent was removed the residue was purified by chromatography(20-60% EtOAc/Hexane) to get the product as a color less liquid (1.12 g,75%). 1HNMR (DMSO-d6) d=MS calculated for C₆₃H₆₂N₄O₁₆ 1130.42. Found1153.40 (M+Na).

Synthesis of 103:

Compound 98 (0.300 g, 1.015 mmol) and the Linolel azide 103 (0.383 g,1.015 mmol) were taken in MeOH/DCM (20 mL, 2:1) mixture and stirred thereaction mixture under argon. Reaction was monitored by TLC; in 4 hrsreaction was complete. Solvent was removed the residue was purified bychromatography (30-60% EtOAc/Hexane) to get the product as a color lessliquid (0.630 g, 92%). 1HNMR (DMSO-d6) d=MS calculated for C₄₁H₅₆N₄O₄Found: 668.43. Found 669.43 (M+H).

Example 3 Synthesis of Solid Support and Amidite

Synthesis of Compound 106:

To a stirred solution of compound 98 (5.00 g, 13.24 mmol), 105 (8.45 g,15.88 mmol) and DIEA (6.90 mL, 3 eq) in DMF (100 mL); HBTU (6.41 g, 1.3eq) was added and stirred the solution overnight at room temperature.The mixture was poured in to an ice water mixture as extracted withEthyl acetate. Dried over sodium sulfate and the crude product werepurified by chromatography (3-5% MeOH/DCM) to get the product as paleyellow solid (9.87 g, 87%). MS calculated for C₅₅H₆₁N₃O₈ 891.45. Found892.40.

Synthesis of Compound 107:

Compound 106 (2.04 g, 2.280 mmol) was dissolved in DCM (20 mL) to thatsuccinic anhydride (0.459 g, 2 eq.) and DMAP (0.850 g, 3 eq.) were addedand stirred the reaction mixture overnight. Solvents were removed andthe residue filtered through a small pad of silica gel. Crude productwas used for the next reaction. The above compound dissolved in DMF (100mL) to that HBTU (1.29 g, 1.5 eq) and DIEA (1.19 mL, 3 eq.) were addedswirl for few minutes. Solid supports (Alkyl amino CPG, 22 g) was addedand shake the mixture for 4 hrs. Filtered, washed with DCM, DCM/MeOH andanhydrous ether. Solid support was capped with acetic anhydride andpyridine. Repeated the same washing process and dried the support undervacuum (23.5 g, 59.50 mol/g).

Synthesis of Compound 108:

Compound 106 (3.00 g, 3.36 mmol) was dissolved in DCM (30 mL) to thatDIEA (1.16 mL, 2 eq.) and Chloroamidite reagent were added and stirredthe mixture for 30 minutes at room temperature. Reaction was monitoredby TLC, reaction mixture was transferred to a separatory funnel washedwith water and sodium bicarbonate solution. Crude product was purifiedby chromatography (EtOAc/Hexane) to get the compound as white fluffysolid (2.93 g, 79%).

Example 4 Synthesis of Single-Stranded RNA Containing Active Alkyne bySolid Phase Method

RNA oligonucleotides containing 3′, internal, and 5′ alknye (Table 1-4)were synthesized on a DNA synthesizer ABI 394 using standardphosphoramidite chemistry with commercially available5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite monomers of uridine (U), 4-N-benzoylcytidine (C^(Bz)),6-N-benzoyladenosine (A^(Bz)) and 2-Nisobutyrylguanosine (G^(iBu)) with2′-O-t-butyldimethylsilyl protected phosphoramidites, and5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (T). Alkyne phosphoramidte (Q99) and alkyne-CPG (L146)used in this study are shown in Table 1 and Table 2. After cleavage andde-protection of part of RNA products, RNA oligonucleotides werepurified by reverse phase high-performance liquid chromatography(RP-HPLC) and characterized by LC-MS.

Example 5 Cu(I)-Free Click Reactions of 5′-alkyne-RNA (A53215.1) withGalNAc (Protected), Mannose (Protected) and C18 Azides in Solution Phase(FIG. 1)

To 5′-alkyne-RNA (A53215.1) (0.05 μmol, 89 μL RNA from 0.56 mM stocksolution in water) was added an azide (FIG. 1) (20 equiv by alkyne, 1μmol, 20 μL azide of a 50 mM solution in methanol for GalNAc (protected)azide and mannose (protected) azide, THF for C18 azide. MeOH was addedto obtain a total volume of 200 μL for click reactions with GalNAc(protected) azide. For mannose (protected) azide, MeOH/THF (1:1 v/v) wasadded to obtain a clear solution of 300 μL. For click reaction with C18azide, MeOH/THF (1:1 v/v) was added to obtain a clear solution of 200μL. After mixing, the click reaction was immediately monitored at roomtemperature by analytical RP-HPLC by directly injecting 1 μL reactionmixture into an Agilent HPLC with DNAPAC™ PA-200 column (4×250 mm) and agradient of 8-20% buffer B in 16 min at a flowrate of 1 mL/min. Buffer Acontains 20 mM Tris HCl pH 8.0, 10 mM NaClO₄, 1 mM EDTA and 50% ACN.Buffer B contains 20 mM Tris HCl, pH 8.0, 800 mM NaClO₄, 1 mM EDTA and50% ACN. The HPLC analysis is shown in FIG. 2.

It is shown that click reaction of 5′-alkyne-RNA (A53215.1) with GalNAc(protected) azide is very fast. After one hour, the reaction iscompleted (FIG. 2a &d). The click reaction of 5′-alkyne-RNA (A53215.1)with mannose (protected) azide is a little slower, but after five hour,the reaction is completed (FIG. 2b &d). The click reaction of5′-alkyne-RNA (A53215.1) with C18 azide is the slowest. After one hour,there is no reaction detected by RP-HPLC. After 3 hours, there is only38% product generated. After 20 hours, the reaction is completed (FIG.2c &d). All click products were confirmed with right molecular weight byLC/MS analysis (Table 1).

Example 6 Cu(I)-Free Click Reactions of 5′-alkyne-RNA (A53215.1) withGalNAc3 (Unprotected) and Mannose (Unprotected) in Solution Phase (FIG.1)

To 5′-alkyne-RNA (A53215.1) (0.05 μmol, 89 μL RNA from 0.56 mM stocksolution in water) was added an azide (FIG. 1) (20 equiv by alkyne, 1μmol, 20 μL azide of a 50 mM solution in methanol for GalNAc3(unprotected) azide and mannose (unprotected) azide. MeOH was added toobtain a total volume of 200 μL. After mixing, the click reaction wasimmediately monitored at room temperature by analytical RP-HPLC bydirectly injecting 1 μL reaction mixture into an Agilent HPLC withDNAPAC™ PA-200 column (4×250 mm) and a gradient of 8-20% buffer B in 16min at a flowrate of 1 mL/min. Buffer A contains 20 mM Tris HCl pH 8.0,10 mM NaClO₄, 1 mM EDTA and 50% ACN. Buffer B contains 20 mM Tris HCl,pH 8.0, 800 mM NaClO₄, 1 mM EDTA and 50% ACN. The HPLC analysis is shownin FIG. 3.

It is shown that click reaction of 5′-alkyne-RNA (A53215.1) with GalNAc3(unprotected) azide is moderately fast. After one hour, the reaction ismore than 80% completed (FIG. 3a &c). After 3 hours, the reaction wasalmost 100% completed. The click reaction of 5′-alkyne-RNA (A53215.1)with mannose (protected) azide is very fast in this study. After onehour, the reaction is completed (FIG. 3b &c). All click products wereconfirmed with right molecular weight by LC/MS analysis (Table 1). Theimpurity peak observed in the click reaction of 5′-alkyne-RNA (A53215.1)with GalNAc3 (unprotected) azide is expected from azide monomer. Themajor impurity has a mass of 8742.01 (−203 compared with the productmass 8945.57) (data not shown).

Example 7 Cu(I)-Free Click Reactions of 3′-alkyne-RNA (A53213.1) withGalNAc (Protected), Mannose (Protected) and C18 Azides in Solution Phase(FIG. 4)

To 3′-alkyne-RNA (A53213.1) (0.05 μmol, 75 μL RNA from 0.67 mM stocksolution in water) was added an azide (FIG. 4) (20 equiv by alkyne, 1μmol, 20 μL azide of a 50 mM solution in methanol for GalNAc (protected)azide and mannose (protected) azide, THF for C18 azide. MeOH was addedto obtain a total volume of 200 μL for click reactions with GalNAc(protected) azide. For mannose (protected) azide, MeOH/THF (1:1 v/v) wasadded to obtain a clear solution of 300 μL. For click reaction with C18azide, MeOH/THF (1:1 v/v) was added to obtain a clear solution of 200μL. The reaction was kept at room temperature for 18 hours or 60 hours.The click reaction was monitored by analytical RP-HPLC by injecting 20μL of 30 fold diluted reaction mixture into an Agilent HPLC with DNAPAC™PA-200 column (4×250 mm) and a gradient of 8-20% buffer B in 16 min at aflowrate of 1 mL/min. Buffer A contains 20 mM Tris HCl pH 8.0, 10 mMNaClO₄, 1 mM EDTA and 50% ACN. Buffer B contains 20 mM Tris HCl, pH 8.0,800 mM NaClO₄, 1 mM EDTA and 50% ACN. The HPLC analysis is shown in FIG.5.

It is shown that click reactions of 3′-alkyne-RNA (A53213.1) with GalNAc(protected), mannose (protected) azide and C18 azide all went completionin this experiment (FIG. 5). All click products were confirmed withright molecular weight by LC/MS analysis (Table 2).

Example 8 Cu(I)-Free Click Reactions Of Internal-Alkyne-RNA (A53214.1)with GalNAc (Protected), Mannose (Protected) and C18 Azides in SolutionPhase (FIG. 6)

To internal-alkyne-RNA (A53214.1) (0.05 μmol, 33 μL RNA from 1.51 mMstock solution in water) was added an azide (FIG. 6) (20 equiv byalkyne, 1 μmol, 20 μL azide of a 50 mM solution in methanol for GalNAc(protected) azide and mannose (protected) azide, THF for C18 azide. MeOHwas added to obtain a total volume of 200 μL for click reactions withGalNAc (protected) azide. For mannose (protected) azide, MeOH/THF (1:1v/v) was added to obtain a clear solution of 300 μL. For click reactionwith C18 azide, MeOH/THF (1:1 v/v) was added to obtain a clear solutionof 200 μL. The reaction was kept at room temperature for overnight (18hours). The click reaction was monitored by analytical RP-HPLC byinjecting 20 μL of 30 fold diluted reaction mixture into an Agilent HPLCwith DNAPAC™ PA-200 column (4×250 mm) and a gradient of 8-20% buffer Bin 16 min at a flowrate of 1 mL/min. Buffer A contains 20 mM Tris HCl pH8.0, 10 mM NaClO₄, 1 mM EDTA and 50% ACN. Buffer B contains 20 mM TrisHCl, pH 8.0, 800 mM NaClO₄, 1 mM EDTA and 50% ACN. The HPLC analysis isshown in FIG. 7.

It is shown that click reactions of internal-alkyne-RNA (A53214.1) withGalNAc (protected), mannose (protected) azide and C18 azide all wentcompletion in this experiment (FIG. 7). All click products wereconfirmed with right molecular weight by LC/MS analysis (Table 3).

Example 9 Cu(I)-Free Click Reactions of 5′-alkyne-RNA (A53215.1) withGalNAc (Protected) and C18 Azides on Solid Support (FIG. 8)

To a solid-supported 5′-alkyne-RNA (A53215.1) in Table 4 (0.646 μmol)was added an azide (15 equiv by alkyne, 10 μmol, 200 μL of a 50 mMsolution in methanol for GalNAc (protected) azide and in THF for C18azide (FIG. 8). The reaction was kept at room temperature for overnight(18 hours). The CPG was filtered, washed and deprotected. The mixtureafter deprotection was analyzed by RP-HPLC on an Agilent HPLC withDNAPAC™ PA-200 column (4×250 mm) and a gradient of 8-20% buffer B in 16min at a flowrate of 1 mL/min. Buffer A contains 20 mM Tris HCl pH 8.0,10 mM NaClO₄, 1 mM EDTA and 50% ACN. Buffer B contains 20 mM Tris HCl,pH 8.0, 800 mM NaClO₄, 1 mM EDTA and 50% ACN. The HPLC analysis is shownin FIG. 9.

It is shown that click reactions of 5′-alkyne-RNA (A53215.1) with GalNAc(protected), and C18 azide on solid support both went completion in thisexperiment (FIG. 9). All click products were confirmed with rightmolecular weight by LC/MS analysis (Table 4).

TABLE 1 Click 5′-alkyne-RNA with azides in solution phase MW Calc. MWSequence ID Note (g/mol) obs. (g/mol) A53215.1 5′-alkyne-RNA, startingmaterial 7257.67 7256.77 A53215_Gal Click with GalNAc (protected)7762.16 7761.45 azide A53215_Man Click with mannose (protected) 8011.428010.55 azide A53215_C18 Click with C18 azide 7549.14 7548.42A53215_GalDep Click with GalNAc3 8945.59 8945.57 (unprotected) azideA53215_ManDep Click with mannose (unprotected) 7595.03 7592.94 azideNote: A-53215.1 sequence (5′-3′) information: Q99CUUACGCUGAGUACUUCGAdTdT(SEQ ID NO: 32)

TABLE 2 Click 3′-alkyne-RNA with protected azides in solution phase MWMW Calc. obs. Sequence ID Note (g/mol) (g/mol) A53213.1 3′-alkyne-RNA,starting material 7258.68 7256.73 A53213_Gal Click with GalNAc(protected) azide 7763.17 7761.44 A53213_Man Click with mannose(protected) azide 8012.43 8010.56 A53213_C18 Click with C18 azide7550.15 7548.41 Note: A-53213.1 sequence (5′-3′) information:CUUACGCUGAGUACUUCGAdTdTL146 (SEQ ID NO: 33)

TABLE 3 Click internal-alkyne-RNA with protected azides in solution phase MW MW Sequence Calc. obs. ID Note (g/mol) (g/mol)A53214.1 Internal-alkyne-RNA,  6951.5 6950.72 starting materialA53214_Gal Click with GalNAc  7455.99 7455.43 (protected) azideA53214_Man Click with mannose  7705.25 7704.54 (protected) azideA53214_C18 Click with C18 azide 7242.97 7242.51 Note: A-53214.1 sequence(5′-3′) information: CUUACGCUGAGQ99ACUUCGAdTdT (SEQ ID NO: 34) (Q99refers to Table 3)

TABLE 4 Click 5′-alkyne-RNA-CPG with protected azides  on solid supportMW  MW  Sequence Calc. obs. ID Note (g/mol) (g/mol) A53215.15′-alkyne-RNA, starting  7257.67 7256.77 material 215CPG_GalClick with GalNAc   7636.08 7635.22 (protected) azide on CPG 215CPG_C18Click with C18 azide  7549.14 7547.49 on CPG Note: A53215.1 sequence(5′-3′) information: Q99CUUACGCUGAGUACUUCGAdTdT-CPG (SEQ ID NO: 32) (Q99refers to Table 3)

Synthesis of New Alkyne Derivatives

TABLE 5 Alkyne derivatives Structure 11

22

33

44

55

66

77

88

99

110

111

112

Example 10 Synthesis Alkyne Derivative 508

Alkyne derivative 508 is prepared by following the procedure given inscheme 1

Example 11 Synthesis of Alkyne Derivative 517

Alkyne derivative 517 is prepared by following the procedure given inscheme 1

Example 12 Synthesis of alkyne derivative 529

Alkyne derivative 529 is prepared by following the procedure given inscheme 1

Example 13 Synthesis of Alkyne Derivative 538

Alkyne derivative 538 is prepared by following the procedure given inscheme 1

Example 14 Synthesis of Alkyne Derivative 547

Alkyne derivative 547 is prepared by following the procedure given inscheme 1

Example 15 Synthesis of Alkyne Derivative 556

Alkyne derivative 556 is prepared by following the procedure given inscheme 1

Example 16 Synthesis of Alkyne derivative 565

Alkyne derivative 565 is prepared by following the procedure given inscheme 1

Example 17 Synthesis of Alkyne Derivatives 576

Alkyne derivative 567 is prepared by the Diels alder reaction of furanor thiophene with corresponding acetylene derivatives.

Example 18 Synthesis of 2′ and 3′ Amino Derivatives

(For the synthesis of 2′ and 3′ phthalimido derivatives follows NucleicAcids Symposium Series No. 52 51-52).

Example 19 Synthesis of 2′ Alkyne Derivative 709

Synthesis of compound 709: Compound 704 is treated with methyl amine (33wt % in ethanol) overnight to get compound 707. This derivative iscoupled with the alkyne derivative 98 using HBTU/DIEA to get thehydroxyl compound 708. This is loaded on to the solid support usingmethod described in scheme 3.

Example 20 Synthesis of 3′ Alkyne Derivative 712

Synthesis of compound 712: Compound 703 is treated with methyl amine (33wt % in ethanol) overnight to get compound 710. This derivative iscoupled with the alkyne derivative 98 using HBTU/DIEA to get thehydroxyl compound 711. Amidite derivative 712 is synthesized usingmethod described in scheme 3.

Example 21 Synthesis of 5′ Alkyne Derivative 719

Synthesis of compounds 718 & 719: Compound 713 is treated withphthalimido protected diamine in presence of DSC and TEA to get 714.Phthalimido group is deprotected with methyl amine at RT to get 715.This compound is treated with the alkyne derivative to obtain thehydroxyl derivative 716. From this compound both the amidite and solidsupport is synthesized by the methods described earlier.

Example 22 Synthesis of Cytidine Derivatives

Scheme 16 R R′

 

 

 

 

(For the synthesis of compound 720 follows Manoharan M. Designerantisense oligonucleotides: Conjugation chemistry and Functionalityplacement, Chapter 17, Antisense research and applications Crooke, S.T.; and Lebleu, B. 1993 CRC and Manoharan M. Antisense & Nucleic acidDrug development 2002, 12, 103-128 and references there in)

Example 23 Synthesis of C-5 Derivatives

Scheme 17 R R′

 

 

 

 

(For the synthesis of compound 720 follows Manoharan M. Designerantisense oligonucleotides: Conjugation chemistry and Functionalityplacement, Chapter 17, Antisense research and applications Crooke, S.T.; and Lebleu, B. 1993 CRC and Manoharan M. Antisense & Nucleic acidDrug development 2002, 12, 103-128 and references there in)

Example 24 Synthesis of RNA Conjugates

Example 25 Synthesis of RNA Conjugates

Example 25 Pseudouridine Conjugates

Synthesis of Polymer-siRNA Conjugates Through Metal Free Click Chemistry

Metal free click chemistry is used making different conjugatescontaining HPMA, polypropyl acrylic acid derivatives, polyketal andother endo-osmolytic polymers with siRNA either in the 3′ or 5′ end.siRNA can be conjugated to targeting ligands on 3′ or 5′ end.

Example 26 Synthesis of Azide Group Containing HPMA Copolymer and itsConjugation to Alkyne Functionalized siRNA and Endosomolytic Group

Example 26 Synthesis and Conjugation of HPMA Copolymer ContainingNitrile Oxides and Conjugation with Alkynes by Metal Free ClickChemistry

Polymer Characterization

The polymers are characterized for their composition by NMR. Themolecular weights (number average molecular weight and weight averagemolecular weight) and the polydispersity of the polymers are determinedby gel permeation chromatography (GPC) coupled with a Multi Angle LaserLight Scattering (MALLS) instrument and a Refractive index (RI)detector. The determined values will be the absolute ones which are notbased on polymer standards. The hydrodynamic radii of the polymers aredetermined from the viscosity detector and light scattering instrument.The size measurements will also be measured using a dynamic lightscattering instrument.

Example 27 Synthesis of a Copolymer of HPMA,N-(3-azidopropyl)methacylamide) and GalNAc₃-methacrylamide

Copolymers are prepared by solution radical copolymerization in DMSO at60 OC using AIBN (1 wt. %) as initiator and monomers (14 wt. %).N-(3-azidopropyl)methacylamide) is synthesized by the reaction of1-Azido-3-aminopropane and methacryloyl chloride (Huang, C and Chang F.Macromolecules 2009, 42, 5155-5166). The monomers 100, 101 and 102 (1,0.25 and 0.125 mmol) mixed with AIBN are dissolved in DMSO, bubbled withargon for 3 min and polymerized in a high-pressure-resistant ampoule at60 OC for 6 hrs. The crude copolymer is precipitated in dryacetone-ether mixture (1:3, 100 ml). The product is re-precipitated fromdry acetone-diethyl ether mixture (3:1, 500 ml), filtered and driedunder vacuum.

Example 28 Synthesis of a Copolymer of HPMA, 3,3′-diethoxypropylmethacrylate and GalNAc₃-methacrylamide

3,3′-diethoxypropyl methacrylate (809) is prepared by reported procedure(J. Zabransky, M. Houska and J. Kalal, Makromolekulare Chemie.Macromolecular Chemistry and Physics 186 (2) (1985), pp. 223-229). Themonomers 100, 109 and 102 (1, 0.25 and 0.125 mmol) mixed with AIBN aredissolved in DMSO, bubbled with argon for 3 min and polymerized in ahigh-pressure-resistant ampoule at 60° C. for 6 hrs. The crude copolymeris precipitated in dry acetone-ether mixture (1:3, 100 ml). The productis re-precipitated from dry acetone-diethyl ether mixture (3:1, 500 ml),filtered and dried under vacuum.

Example 29 Conjugation of siRNA to the Polymer 812

To the alkyne functionalized siRNA (1 mmol) dissolved in ethanol and 4%aqueous NaHCO3 is added the polymer oxime 112 (1 mmol) and chloramines-T(2 mmol). The mixture was stirred at room temperature for 16 hrs. Thereaction mixture was purified by dialysis against water and freeze dried

REFERENCES

-   Rutjes, Floris Petrus Johannes Theodorus; Cornelissen, Johannes    Lambertus Maria; Van Berkel, Sander Sebastiaan; Dirks, Antonius    Johannes. Process for preparation of trisubstituted 1,2,3-triazoles.    PCT Int. Appl. (2008), 63 pp. CODEN: PIXXD2 WO 2008075955 A2    20080626-   van Berkel, Sander S.; Dirks, A. J.; Debets, Marjoke F.; van Delft,    Floris L.; Cornelissen, Jeroen J. L. M.; Nolte, Roeland J. M.;    Rutjes, Floris P. J. T. Metal-free triazole formation as a tool for    bioconjugation. ChemBioChem (2007), 8(13), 1504-1508.-   A procedure for fast and regioselective copper-free click chemistry    at room temperature with p-toluenesulfonyl alkyne. Gouin, Sebastien    G.; Kovensky, Jose. Department of Chemistry, Laboratoire des    Glucides UMR CNRS 6219, Institut de Chimie de Picardie, Universite    de Picardie Jules Verne, Amiens, Fr. Synlett (2009), (9), 1409-1412.-   Fast, copper-free click chemistry: A convenient solid-phase approach    to oligonucleotide conjugation. Singh, Ishwar; Vyle, Joseph S.;    Heaney, Frances. Department of Chemistry, National University of    Ireland, Co. Kildare, Maynooth, UK. Chemical Communications    (Cambridge, United Kingdom) (2009), (22), 3276-3278.-   Bertozzi, Carolyn Ruth; Agard, Nicholas J.; Prescher, Jennifer A.;    Baskin, Jeremy Michael; Sletten, Ellen May. Preparation of    cyclooctynes and azacyclooctynes for modification of biomolecules in    vivo and in vitro by their copper-free strain-promoted [3+2]    cycloaddition with azides. U.S. Pat. Appl. Publ. (2009), 62 pp.,    Cont.-in-part of U.S. Ser. No. 264,463. CODEN: USXXCO US 2009068738    A1 20090312 CAN 150:330128 AN 2009:291769-   De Forest, Cole A.; Polizzotti, Brian D.; Anseth, Kristi S.    Sequential click reactions for synthesizing and patterning    three-dimensional cell microenvironments. Nature Materials (2009),    8(8), 659-664.-   Singh, Ishwar; Zarafshani, Zoya; Lutz, Jean-Francois; Heaney,    Frances. Metal-Free “Click” Chemistry: Efficient Polymer    Modification via 1,3-Dipolar Cycloaddition of Nitrile Oxides and    Alkynes. Macromolecules (Washington, D.C., United States) (2009),    42(15), 5411-5413.

1. A compound of formula I, or pharmaceutically acceptable thereof:

wherein: X is O, S, NR^(N) or CR^(P) ₂; B is independently for eachoccurrence hydrogen, optionally substituted natural or non-naturalnucleobase, optionally substituted triazole or optionally substitutedtetrazole; NH—C(O)—O—C(CH₂B₁)₃, NH—C(O)—NH—C(CH₂B₁)₃; where B₁ ishalogen, mesylate, N₃, CN, optionally substituted triazole or optionallysubstituted tetrazole; R¹, R², R³, R⁴ and R⁵ are each independently foreach occurrence H, OR⁶, F, N(R^(N))₂, N₃, CN, -J-linker-N₃,-J-linker-CN, -J-linker-C≡R⁸, -J-linker-cycloalkyne, -J-linker-R^(L),-J-Q-linker-R^(L), -J-linker-Q-R^(L), -J-linker-Q-linker-R^(L),-J-linker-J-Q-R^(L), or -J-linker-J-Q-linker-R^(L); J is independentlyfor each occurrence absent, O, S, NR^(N), OC(O)NH, NHC(O)O, C(O)NH,NHC(O), NHSO, NHSO₂, NHSO₂NH, OC(O), C(O)O, OC(O)O, NHC(O)NH, NHC(S)NH,OC(S)NH, O—N═CH, OP(N(R^(N))₂)O, or OP(N(R^(N))₂); R⁶ is independentlyfor each occurrence hydrogen, hydroxyl protecting group, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedcycloalkyl, optionally substituted aralkyl, optionally substitutedalkenyl, optionally substituted heteroaryl, polyethyleneglycol (PEG), aphosphate, a diphosphate, a triphosphate, a phosphonate, aphosphonothioate, a phosphonodithioate, a phosphorothioate, aphosphorothiolate, a phosphorodithioate, a phosphorothiolothionate, aphosphodiester, a phosphotriester, an activated phosphate group, anactivated phosphite group, a phosphoramidite, a solid support,—P(Z¹)(Z²)—O-nucleoside, —P(Z¹)(Z²)—O-oligonucleotide,—P(Z¹)(Z²)-formula (I), —P(Z¹)(O-linker-Q-linker-R^(L))—O-nucleoside,P(Z¹)(O-linker-R^(L))—O-nucleoside, —P(Z¹)(O-linker-N₃)—O-nucleoside,P(Z¹)(O-linker-CN)—O-nucleoside, P(Z¹)(O-linker-C≡R⁸)—O-nucleoside,P(Z¹)(O-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(O-linker-Q-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-R^(L))—O-oligonucleotide,P(Z¹)(O-linker-N₃)—O-oligonucleotide,—P(Z¹)(O-linker-CN)—O-oligonucleotide,P(Z¹)(O-linker-C≡R⁸)—O-oligonucleotide,P(Z¹)(O-linker-cycloalkyne)-O-oligonucleotide,—P(Z¹)(-linker-Q-linker-R^(L))—O-nucleoside,—P(Z¹)(-linker-Q-R^(L))—O-nucleoside, —P(Z¹)(-linker-N₃)—O-nucleoside,P(Z¹)(-linker-CN)—O-nucleoside, P(Z¹)(-linker-CR⁸)—O-nucleoside,P(Z¹)(-linker-cycloalkyne)-O-nucleoside,—P(Z¹)(-linker-Q-linker-R^(L))—O-oligonucleotide,—P(Z¹)(-linker-R^(L))—O-oligonucleotide,P(Z¹)(-linker-N₃)—O-oligonucleotide,—P(Z¹)(-linker-CN)—O-oligonucleotide,P(Z¹)(-linker-C≡R⁸)—O-oligonucleotide orP(Z¹)(-linker-cycloalkyne)-O-oligonucleotide; R^(N) is independently foreach occurrence H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted cycloalkyl, optionally substituted aralkyl,optionally substituted heteroaryl or an amino protecting group; R^(P) isindependently for each occurrence H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted cycloalkyl oroptionally substituted heteroaryl; Q is absent or independently for eachoccurrence

X₁ is O, S, CF₂, or CH₂; Y₁, Y₂ and Y₃ are each independent CRP, N, O,or S; W₁ is CH or N; R₁₀₀, R₂₀₀, R₃₀₀ and R₄₀₀ are each independentlyhydrogen, halogen, OR^(N), CR^(P) ₂, acyl, phosphonyl, sulfonyl; oralternatively, R₁₀₀ and R₂₀₀ or R₃₀₀ and R₄₀₀ are taken together to forman aryl, substituted aryl, heteroaryl, substituted heteroaryl,cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substitutedheterocycloalkyl; R^(L) is hydrogen or a ligand; R⁸ is N or CR⁹; R⁹ isH, optionally substituted alkyl or silyl; and Z¹ and Z² are eachindependently for each occurrence O, S or optionally substituted alkyl;provided that at least one of R¹, R², R³, R⁴ and R⁵ is-J-linker-Q-R^(L), -J-linker-Q-linker-R^(L), -J-linker-J-Q-R^(L), or-J-linker-J-Q-linker-R^(L).
 2. The compound of claim 1, represented byformula (VI)

wherein each of R₅₀₀ is independently hydroxyl, halogen, acyl, sulfonyl,phosphonyl, alkyl, haloalkyl, NO₂, CN, N₃, alkoxy, aminoalkyl,aminodialkyl, and thioalkyl; n is 0-4; each linker can be the same ordifferent; and B, R³, R⁵, R^(L), J, and X are as defined in claim
 1. 3.The compound of claim 1, represented by formula (VIa)

wherein Y₁-Y₆ are each independently O, S, NR^(N), or CR^(P) ₂; eachlinker can be the same or different; and B, R³, R⁵, R^(L), R^(N), R^(P),J, and X are as defined in claim
 1. 4-8. (canceled)
 9. The compound ofclaim 1, where R^(L) is selected from the group consisting of:


10. The compound of claim 1, wherein each linker is represented bystructure—[P-Q₁-R]_(q)-T-, wherein: P, R and T are each independently for eachoccurrence absent, CO, NH, O, S, S—S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O;NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, —C(O)— (optionally substitutedalkyl)-NH—, CH═N—O,

acetal, ketal,

Q₁ is independently for each occurrence absent, —(CH₂)_(n)—,—C(R¹⁰⁰)(R²⁰⁰)(CH₂)_(n)—, —(CH₂)_(n)C(R¹⁰⁰)(R²⁰⁰)—,—(CH₂CH₂O)_(m)CH₂CH₂—, or —(CH₂CH₂O)_(m)CH₂CH₂NH—; R^(a) is H or anamino acid side chain; R¹⁰⁰ and R²⁰⁰ are each independently for eachoccurrence H, CH₃, OH, SH or N(R^(X))₂; R^(X) is independently for eachoccurrence H, methyl, ethyl, propyl, isopropyl, butyl or benzyl; q isindependently for each occurrence 0-20; n is independently for eachoccurrence 1-20; and m is independently for each occurrence 0-50. 11.The compound of claim 1, where least one of R¹, R², R³, R⁴, and R⁵ offormula (I) is

wherein Y₁, Y₂, Y₃, R₁₀₀, R₂₀₀, R₃₀₀, R₄₀₀, and R^(L) are as previouslydefined in claim
 1. 12. A method of activating target-specific RNAinterference (RNAi) in an organism comprising administering to saidorganism the siRNA or composition of claim 1, said compound beingadministered in an amount sufficient for degradation of the target mRNAto occur, thereby activating target-specific RNAi in the organism. 13.The method of claim 12, wherein the target mRNA specifies the amino acidsequence of a protein involved or predicted to be involved in a humandisease or disorder.
 14. The method of claim 13, wherein the disease ordisorder is selected from the group consisting of viral infections,bacterial infections, parasitic infections, cancers, allergies,autoimmune diseases, immunodeficiencies, and immunosuppression.
 15. Thecompound of claim 1, wherein at least one of R¹, R², R³, R⁴ and R⁵ isOR⁶, wherein R⁶ contains an oligonucleotide.
 16. The compound of claim15, wherein the oligonucleotide is a single-stranded oligonucleotide.17. The compound of claim 16, wherein the single-strandedoligonucleotide is a single-stranded siRNA.
 18. The compound of claim15, wherein the oligonucleotide is a double-stranded oligonucleotide.19. The compound of claim 18, wherein the double-strandedoligonucleotide is a double-stranded siRNA.
 20. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable excipient.